PROTOCOL

Imaging activation of adult-generated granule cells in
spatial memory
Nohjin Kee1–3,5, Cátia M Teixeira1–5, Afra H Wang1,3,5 & Paul W Frankland1–3
1Program in Neurosciences and Mental Health, The Hospital for Sick Children, Toronto, Canada M5G 1X8. 2Department of Physiology, University of Toronto, Toronto,

Canada, M5S 1A8. 3Institute of Medical Science, University of Toronto, Toronto, Canada M5S 1A8. 4Graduate Program in Areas of Basic and Applied Biology (GABBA),
Universidade do Porto, 4050-465 Porto, Portugal.. 5These authors contributed equally to this work. Correspondence should be addressed to P.W.F. (paul.frankland@sickkids.ca).

© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

Published online 21 November 2007; doi:10.1038/nprot.2007.415

New neurons are continuously generated in the subgranular zone of the hippocampus throughout adulthood, and there is increasing
interest as to whether these new neurons become functionally integrated into memory circuits. This protocol describes theimmunohistochemical procedures to visualize the recruitment of new neurons into circuits supporting spatial memory in intact mice.
To label adult-generated granule cells, mice are injected with the proliferation marker 5-bromo-2¢-deoxyuridine (BrdU). At different
delays after BrdU treatment, mice are trained to locate a hidden platform in the Morris water maze, and spatial memory can then be
tested in a probe test with the platform removed from the pool. Ninety minutes after this probe test, mice are perfused and tissue
is sectioned. Immunohistochemical procedures are used to quantify BrdU-labeled cells and expression of the immediate early gene,
Fos. Because Fos expression is regulated by neuronal activity, the degree of overlap between BrdU-labeled and Fos-labeled neurons
provides an indication of whether adult-generated granule neurons have been incorporated into spatial memory circuits.

INTRODUCTION
It is well established that new neurons are continuously generated
in the subgranular zone of the dentate gyrus throughout adulthoodin all mammals1. Within a few days, newly generated neurons
migrate into the granule cell layer, begin to extend dendritic
processes toward the molecular layer and an axon toward theCA3 region. At this stage, immature neurons may receive
g-aminobutyric acid-mediated excitatory synaptic inputs. However, major glutamatergic synaptic activation from perforant pathafferents does not occur until new neurons are at least 2 weeks old,when spines begin to form2. Because the hippocampus plays a keyrole in many forms of memory, there is considerable interest in
how these new neurons might contribute to hippocampal memory
processing3.
One way this issue has been addressed is to examine the effectsof suppressing adult neurogenesis on hippocampal learning. To
suppress adult neurogenesis, the two most common strategies
have been irradiation4 (either whole brain or focal) or administering antimitotic agents such as methylazoxymethanol acetate5.
However, the effects of these manipulations on hippocampal
learning have been mixed6. In some cases these treatments disrupt
hippocampal learning5,7–9, whereas in others these treatments
either have no effect7,9–11 or even enhance some forms of learning12. When these treatments produce effects, it has been difficult torule out nonspecific effects on mature cells or on general health13,14.
The absence of treatment effects are equally difficult to interpret.
Because these treatments typically produce an incomplete knockdown of adult neurogenesis (B80%)13,15, it is difficult to rule out
the possibility that residual adult-generated neurons are sufficient
to support hippocampal learning or whether a sufficiently maturecohort of adult-generated neurons have been eliminated. Even if
these treatments were to completely eliminate adult neurogenesis,
it is still possible that hippocampal learning might be supportedby the existing or mature granule cells3.
An alternative strategy is to use immunohistochemical approaches to visualize the recruitment of adult-generated neurons into

the memory circuits in intact mice16–19. The basic scheme is toinject mice with the proliferation marker 5-bromo-2¢-deoxyuridine(BrdU) to ‘birth-date’ the dividing cell population20–22. Mice aresubsequently trained in the Morris water maze, and spatial memory
is assessed in a probe test with the platform removed from thepool. After this spatial memory test, expression of the immediate
early gene Fos is quantified in order to identify the granule cells
processing spatial memory17. The overlap between BrdU-labeledand Fos-labeled neurons then provides an indication of whether or
not adult-generated granule neurons have been incorporated into
spatial memory circuits. The protocol described here is for studying
the contribution of adult-generated granule cells to water mazememory in mice. However, it could easily be adapted for use in rats,
and with other types of hippocampus-dependent learning tasks.
Water maze
Developed in the early 1980s by Richard Morris23, the water mazehas become one of the most commonly used tasks to measurespatial learning in rodents, including normal and geneticallymodified mice24. In this task, training typically takes place over
several days in a large, circular tank filled with opaque water. In
each training trial, a mouse is given the opportunity to navigate to aplatform submerged below the water surface. Because the platform
is not visible, the mouse must locate it using an array of distal,visual cues surrounding the pool. After the completion of training,spatial memory can then be assessed at different retention delays ina probe test, where the platform is removed from the pool and themouse is allowed to search for it using the distal cues.
For these studies, the water maze offers several important
advantages. First, lesion, pharmacological, genetic and neuroimaging studies have established an essential role for the hippocampus
in the formation of water maze memories25. Second, mice trainedin this task exhibit robust and long-lasting spatial memory. Whilesome previous studies have emphasized species differences in
performance in the water maze26,27, it is worth stressing that

NATURE PROTOCOLS | VOL.2 NO.12 | 2007 | 3033

mice learn this task very well and show robust retention up to
several weeks17,28 (Fig. 1). Third, expression of this memory
remains dependent on the activity in the hippocampus, even atvery long retention delays29. For example, pharmacological inactivation of the dorsal hippocampus blocks the expression of either aday-old or month-old water maze memory28. These latter two
features are particularly advantageous in designing experiments,
since it allows greater flexibility in the use of retention delays
between training and testing.
There are several important methodological issues to consider
in water maze studies. First, water maze performance is highly
dependent on background strain of the mouse30,31. For example,
mice in a pure C57B6 background are much poorer compared tohybrid C57B6/129 mice, requiring more training to produce robust
long-term retention (Fig. 2). Second, anxiety can interfere withwater maze performance. There are several ways to minimize theconfounding effects of anxiety on learning. For example, the poolshould be dimly lit, and mice should be handled extensively before
the commencement of training. In our studies, mice are handled
for 2 min d1 for 7 d.

50

Percent search

40
30
20
10
0
1

14

28

42

56

70

Retention interval (d)Figure 1 | Long-lasting water maze memory in mice. C57B6/129 mice weretrained in the water maze (5 d, six trials d1) and tested 1 d (n ¼ 9), 14 d(n ¼ 9), 28 d (n ¼ 9), 42 d (n ¼ 10), 56 d (n ¼ 10) or 70 d (n ¼ 9) later.
At each retention delay, mice spent significantly more time searching thetarget zone (dark blue) compared to other (light blue) zones, and there waslittle decline in performance even at longer retention delays. Adapted fromKee et al.17.

3034 | VOL.2 NO.12 | 2007 | NATURE PROTOCOLS

Percent search

activation (such as Fos) makes it possible to determine whether a
BrdU-labeled cell has been recently activated. Similarly, as newborn
granule cells sequentially express different markers as they age,
BrdU
BrdU is used almost universally to detect proliferating cells in the double- or triple-labeling makes it possible to determine the state ofstudies of adult neurogenesis20–22. BrdU crosses the blood–brain differentiation and maturation of BrdU-labeled cells. Second, Ki67
immunohistochemistry provides a ‘snapshot’ of proliferating cells
barrier, and therefore can be administered systemically, either byat the time of perfusion34. In contrast, BrdU immunohistocheminjection or, somewhat less commonly, orally (e.g., via drinking
water). As BrdU is a thymidine analog, it competes with endogen- istry identifies cells that were undergoing division at the time of
injection. By varying the delay between BrdU treatment and waterous thymidine for incorporation into dividing cells during S phase(DNA synthesis) of the cell cycle. Incorporation into the DNA of maze training, it is therefore possible to assess the incorporation of
the cell is permanent, and BrdU may be detected weeks, months or different aged cohorts of BrdU-labeled cells into memory circuits.
even years later in fixed tissue using specific primary antibodies32. Retroviral GFP approaches also make it possible to ‘birth-date’ cells
For visualization, the primary antibody may either be labeled with undergoing division. However, retroviruses must be microinfuseda secondary antibody tagged with a fluorescent compound or with directly into the dentate gyrus (rather than administered systemically) and are therefore more labor-intensive. Furthermore, retroan enzyme for diaminobenzidine as a substrate for visualization.
Until the mid-1990s, radiolabeled thymidine (3H-thymidine) viral approaches typically label fewer cells compared to BrdU, andwas used to label dividing cells33. In addition, more recent studies GFP expression may be silenced over time35,36. Therefore, current
have used endogenous cell-cycle markers, such as Ki67 (ref. 34) and retroviral-based approaches may not be optimal for these sorts of
GFP-expressing retroviruses35, to identify dividing cells. However, studies, but may be particularly suited to more detailed morphofor this protocol the application of BrdU offers two chief advan- logical35,37 and electrophysiological38–40 analyses.
There are several important methodological issues to consider
tages over these techniques. First, unlike 3H-thymidine, immunohistochemical methods are used to detect incorporated BrdU. when using BrdU. First, what dose of BrdU to use? In the young
Therefore, immunostaining for markers associated with neuronal adult mouse, several thousand new neurons are generated daily,
and, of these, only a small proportion willsurvive beyond 4 weeks41 (Fig. 3). Since
Figure 2 | Comparison
50
60
a
b
the
total number of neurons in the mouseof C57B6/129 and50
dentategyrus is between 239,000 and
C57B6 mice in the40
C57B6/129
water maze. (a) C57B6/
351,000
depending on the strain42, this
40
C57B6
30
129 (n ¼ 9) and C57B6
represents a relatively small subpopulation30(n ¼ 19) mice wereand so BrdU treatment protocols that maxi20
trained in the water
20
mize labeling are preferred. In mice, BrdU is1maze (six trials d ) for10
typically injected at doses of 50–200 mg
10
5 and 7 d, respectively.
kg1. While doses at the higher end of thisThe mean daily escape00latencies (± s.e.m.) for
C57B6/129 C57B6
range have been associated with develop1
2
3
4
5
6
7
Training day
C57B6/129 (openmental abnormalities when given to neocircles) and C57B6
natal mice (e.g., o4 weeks in age)43,44, such(closed circles) are plotted. C57B6/129 mice acquired the task more rapidly. (b) Thirty days after theeffects are not observed in adult mice.
completion of training, mice were given a probe test. In the probe test both C57B6/129 and C57B6
We have found that labeling saturates atmice searched selectively, each spending significantly more time searching the target zone (dark blue)
B200 mg kg1 in adult C57B6/129 mice,compared to other (light blue) zones. However, C57B6/129 spent significantly more time in theand so we recommend use of this dose.
target zone compared to C57B6 mice.
Latency (s)© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

PROTOCOL© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

PROTOCOL

Fos expression
Neuronal stimulation is associated with increases in intracellular
calcium levels through N-methyl D-aspartate (NMDA) receptor
activation or voltage-gated calcium channels. Because theseincreases in calcium lead to the rapid upregulation of immediate
early genes such as Fos, measuring Fos levels allows for the detectionof recently activated neurons53. For example, after stimulation, Fosprotein may be detected immunohistochemically in fixed tissue
using specific primary antibodies, and Fos-positive nuclei can be
subsequently quantified visually or using automated software suchas Image J (National Institutes of Health). Because these immunohistochemical methods offer single-cell resolution, double-labelingfor BrdU makes it possible to determine whether adult-generated
neurons have been recently activated.
Fos has been used as an activity marker in many different
experimental situations. For example, Fos is induced in the
hippocampus after seizures54–56, NMDA-dependent long-term
potentiation (LTP) induction57–59, noxious stimulation60, as well
as after learning61,62.
When using Fos expression to map circuits in behavioral
studies, there are several important issues to consider. First, after

700
600
500
BrdU+ cells

BrdU is usually administered as a series of i.p. injections over thecourse of several days. The chief advantage of this approach is thatthe multiple injections ensure that sufficiently large numbers ofcells are labeled for quantitative analyses. The disadvantage of thisapproach is that temporal resolution is compromised. However,maturation of new neurons to a stage where they begin tocontribute to mnemonic function in the dentate gyrus takes
weeks, rather than days, and so this degree of temporal resolution
is appropriate17.
A second concern is the specificity of BrdU labeling. Because
BrdU is a thymidine analog, in principle it could also be incorporated into any cell undergoing DNA repair, in addition to thosein mitosis45. However, whereas mitosis involves the synthesis of
the entire genome, DNA repair typically involves the synthesisof relatively few nucleotides (e.g., o100). Because differences insynthesis rates are several orders of magnitude, these two processes
should always be easy to discriminate. Consistent with this, several
observations suggest that this risk of false-positive labeling associated with DNA repair is negligible. First, after BrdU treatment,
BrdU-labeled cells are not found throughout the CNS, but rather
are confined to the two major neurogeneic regions in the adultbrain. This specificity is evident even when high doses of BrdU
are used (4400 mg kg1)46,47. Second, even under conditions that
promote inflammation and apoptosis (e.g., irradiation), BrdUlabeling is limited to dividing cells48.
A third major issue is that adult neurogenesis is highly regulated
by a large number of environmental variables. Since recruitment of
new neurons into memory circuits might be influenced by theavailability of new neurons, it is important to ensure that different
experimental groups have equivalent levels of BrdU-labeled neurons. In particular, both proliferation and survival are affected byexercise49, enrichment50 and stress51, and so ensuring that thesefactors are held constant across conditions is essential in these typesof studies. Another major influence on adult neurogenesis is age. As
animals age, the generation of new neurons falls exponentially52
and therefore it is important that animals in different experimental
conditions are age-matched.

400
300
200
100
0
2h

3d

4 wk
6 wk
1 wk
Time post-treatment

8 wk

Figure 3 | Survival of adult-generated granule cells. Separate groups of
C57B6/129 mice were given a single injection of 5-bromo-2¢-deoxyuridine(BrdU) (200 mg kg1) and then perfused at different delays (n Z 3 for allgroups). Maximal numbers of BrdU-labeled cells were observed in the mice
perfused 3 d after BrdU treatment, and declined thereafter. This indicates thatthe majority of adult-generated granule cells do not survive beyond 4 weeks.

stimulation, Fos protein levels peak between 1 and 2 h, decaying
thereafter63. While basal levels of Fos are low in unstimulated animals(e.g., home cage), the relatively poor temporal resolution of Fos
expression means that it is critical that the mice remain minimallystimulated both before and after behavioral testing. For example,
extraneous noises or other stimulation that might stress the miceshould be avoided. Perhaps as important, novelty is associated withelevated Fos expression in several brain regions, including the
hippocampus64. Therefore, care should be taken to ensure thatbehavioral procedures on test days match, as much as possible,
those used during training. Even subtle changes in procedures (e.g.,transport, handling, lighting conditions) might elevate Fos levels
above basal conditions. Despite these caveats, we find that Fos
expression in the dentate gyrus remarkably specific after the water
maze testing. In particular, within the dentate gyrus only 0.5–2.5% ofneurons express Fos after behavioral testing17. This matches previous
immediate early gene65 and electrophysiological66 studies, showingthat a similarly small proportion of granule cells are activated during
spatial exploration, and is consistent with the idea that spatial
information is sparsely encoded in the dentate gyrus. Furthermore,
Fos expression is reduced in mice carrying a point mutation in the aCaMKII gene67 that prevents spatial learning (but otherwise does not
disrupt the regulation of Fos)17. This suggests that nonspecificaspects of the test procedures (e.g., swimming, physical exercise,stress or arousal) do not contribute significantly to Fos expression inneurons in the dentate gyrus.
Second, most behavioral studies have focused on the inductionof Fos after learning61,62. This focus on learning-induced changes in
Fos expression is motivated, in part, by the idea that Fos may play a
key role in activity-dependent modification of circuits underlying
memory formation68. Consistent with this, genetic deletion of c-fos
impairs two forms of hippocampal-dependent learning (contextual
and spatial) and the induction of NMDA receptor-mediated LTP69.
However, as Fos is a general neuronal activity marker, it may also be
used to map the circuits activated after memory recall. Using
this approach, recent studies have mapped the circuits activatedafter recall of spatial28,70, aversive64,71, appetitive72, olfactory73,74
and gustatory75 memories. Indeed, in the water maze we havefound that levels of Fos expression in the dentate gyrus are similar
following training and memory probe17. It is therefore likely that
Fos mediates the stabilization of synaptic remodeling after
NATURE PROTOCOLS | VOL.2 NO.12 | 2007 | 3035

PROTOCOL
Figure 4 | Experimental designs. (a) To determine whether new neurons areincorporated into dentate gyrus, the circuits supporting spatial memory
separate groups of mice are treated with 5-bromo-2¢-deoxyuridine (BrdU) andthen trained in the water maze 1, 2, 4, 6 or 8 weeks later. Spatial memory isassessed in a series of three probe tests 10 weeks after BrdU treatment.
Because the BrdU-probe test delay is fixed, this ensures that all BrdU-labeledcells are the same age at the time of testing. (b) To control for age ofmemory, in this design a fixed retention delay of 4 weeks is used in allgroups, while the delay between BrdU treatment and training is varied.

a

10 wk

BrdU
1 wk

BrdU
2 wk

© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

BrdU

reactivation of existing memory circuits, as well as during memory
formation72.
A third concern is that Fos expression may be developmentally
regulated. Therefore, neuronal activity may induce Fos expression
in mature neurons, but not in newborn granule cells. Indeed, while
seizure activity leads to Fos expression in B80% of mature granulecells, this number falls to B50% in 25-d-old adult-generated
granule cells and 0% in 15-d-old adult-generated granule cells16.
The absence of Fos expression in newborn cells might be either
due to an inability of newborn granule cells to express Fos or
because newborn granule cells are not yet completely integrated
into hippocampal circuitry. In memory studies, one way to rule out
the former possibility is to use a design with a fixed delay between
BrdU labeling and memory testing (but varying the timing of the
training)17. This design ensures that all BrdU-labeled cells are thesame age at the time of testing, and therefore any differences in Fos
expression cannot be accounted for developmental stage.
Expression of other immediate early genes such as Activity-regulated cytoskeletal-associated protein (Arc) (also known as Arg3.1) andEgr1 (also known as zif268, Krox-24, NGIF-A, ZENK) is regulated byneuronal activity, and may be used either in addition, or as alternatives, to Fos16–19. Indeed, we have directly compared Fos and Arc
expressions in tissue from the same mice after water maze testing, andfound that these markers give remarkably similar pattern of results.
Expression of Fos and Arc was limited to a similarly small proportionof granule cells after water maze testing, and both methods indicatedthat maximal recruitment of new neurons into spatial memory
circuits does not occur until new neurons are 4 or more weeks inage17. The advantage of using multiple activity markers is that theirexpression relies on potentially different signal transduction cascades.
Therefore, similar patterns of gene expression must reflect general
neuronal activity rather than activation of a specific signaling cascade.
Experimental designs
The basic experimental protocol involves three main experimental
steps: BrdU treatment, water maze training, water maze testing.
Ninety minutes after this last step, mice are perfused; hippocampal
tissue is processed for BrdU and Fos immunohistochemistry, andthe numbers of BrdU+ and Fos+ and BrdU+/Fos+ cells in thedentate gyrus are quantified. The primary experimental manipulation is the delay between BrdU treatment and training. Because
BrdU provides a ‘birth-date’, varying this delay makes it possible tostudy the integration of different aged cohorts of adult-generated
granule cells into spatial memory circuits. The logic is that if BrdUlabeled cells are incorporated into memory networks at the time oftraining, then there should be overlap between BrdU-positive cells
and Fos-positive or Arc-positive cells at the time of testing.
There are several variations on the experimental design (Fig. 4).
In the first design, a fixed delay between BrdU treatment and mouse
perfusion is used, and the delay between BrdU treatment and3036 | VOL.2 NO.12 | 2007 | NATURE PROTOCOLS

4 wk

BrdU

6 wk

BrdU
8 wk

b

4 wk

BrdU
1 wk

BrdU
1 wk

BrdU
6 wk

training varied. Importantly, this ensures that all BrdU-labeled cells
are the same age at the time of testing, and so potential differencesin Fos expression cannot be accounted for in terms of neurochemical maturation. Using this approach, it is therefore possible toevaluate whether week-old granule cells become incorporated into
the circuits supporting spatial memory. The disadvantage of thisdesign is that not only does the delay between BrdU treatment andtraining vary but so does the delay between training and testing.
Since there is some evidence for time-dependent reorganization of
circuits supporting water maze memory within the hippocampus76, a second design uses a fixed retention delay, but varies
the timing of the BrdU treatment with respect to training. This
ensures that the age of the spatial memory is equivalent acrossgroups. An important group in this design is the group in which
training precedes BrdU treatment. Because new neurons are labeledafter training, this group provides an estimate of Fos expression in
BrdU-labeled cells associated with nonmnemonic aspects of theprobe test (such as swimming, physical exercise, stress or arousal).
In our studies, we have identified very few double-labeled cells inthis condition, suggesting that these nonspecific aspects of thetesting procedures do not contribute significantly to Fos expression
in these new neurons17.
Additional analyses and experiments may be used to control for anumber of different factors. First, Fos and BrdU expression may bequantified in the olfactory bulb, the other major neurogenic region in
the adult brain. Since the olfactory bulb would not be expected toplay a major role in spatial memory, the same pattern of results would
not be predicted. Second, nonlearning controls provide another way
to assess the contribution of nonspecific aspects of the testingprocedures on Fos expression in new and existing neurons in thedentate gyrus. Learning impaired mutant mice (e.g., CaMKIIT286A)or pharmacological manipulations that prevent spatial learning

PROTOCOL© 2007 Nature Publishing Group http://www.nature.com/natureprotocols(e.g., NMDA receptor or protein synthesis inhibitors) may also
be used.
Finally, as the hippocampus is involved in both spatial andnonspatial forms of learning, this protocol may easily be adapted
for other hippocampus-dependent tasks (e.g., contextual fear
conditioning, social transmission of food preference, trace eyeblink conditioning, object recognition). In adapting this protocol,
two issues in particular are worth considering. First, training
conditions that produce long-lasting memory (e.g., 44 weeks)
are desirable, since the use of longer retention delays allows for

greater flexibility in experimental design. For example, while
contextual fear memories may last many weeks (or even months),
object recognition memories are typically less enduring. Second,
whereas the hippocampus is permanently involved in the expression of water maze memories28, its role may be more transient inother forms of hippocampus-dependent learning (such as contextual fear and trace eye-blink conditioning77). However, it is clearlyof great theoretical interest whether or not adult-generated granulecells also contribute in a persistent manner to the expression ofthese types of memory.

MATERIALS
REAGENTS. Mice (see REAGENT SETUP). Chloral hydrate (Sigma, cat. no. C-8383) or other approved anesthetic. Sodium phosphate, monobasic anhydrous (NaH2PO4, 1 kg, FW ¼ 120.0;
Sigma, cat. no. S-0751). Sodium phosphate, dibasic anhydrous (Na2HPO4, 1 kg, FW ¼ 142.0; Sigma,
cat. no. S-0876). Normal goat serum (Sigma, cat. no. G9023). Triton X-100 (t-octylphenoxypolyethoxyethanol, 100 ml; Sigma, cat. no.
T-9284). Trisodium citrate dihydrate (FW ¼ 294.10; Sigma, cat. no. W302600). Paraformaldehyde powder (1 kg; Sigma, cat. no. P6148) (see REAGENTSETUP) ! CAUTION Harmful and should be handled with care. Wear proper
protective gear, laboratory coat and weigh under fume hood.
. (+) BrdU (97%; Sigma-Aldrich, cat. no. 858811) (see REAGENT SETUP)! CAUTION A suspected mutagen and should be handled with care. Wear
proper protective gear, laboratory coat and weigh under fume hood.
. Sucrose (EMD, cat. no. SX1075-3) (see REAGENT SETUP). Rat anti-BrdU primary monoclonal antibody (Clone BU1/75,ICR1; Accurate
Chemical & Scientific, cat. no. OBT0030). Optimal dilution ratio 1:500. Rabbit anti-Fos polyclonal primary antibody (e.g., Calbiochem, cat. no.
PC 38). Optimal dilution 1:5,000. Alexa Fluor 488 chicken anti-rat IgG (H + L) (e.g., Invitrogen, cat. no.
A21470) secondary antibody. Optimal dilution ratio 1:1,000. Alexa Fluor 568 goat anti-rabbit/mouse IgG (H + L) (e.g., Invitrogen,cat. no. A11011) secondary antibody. Optimal dilution ratio 1:1,000. Mouse anti-NeuN monoclonal primary antibody (e.g., Chemicon
International, cat. no. MAB377). Optimal dilution 1:1,000. Mounting medium (e.g., Shandon PermaFluor; Thermo Scientific,
cat. no. 434990). PBS (see REAGENT SETUP). Hydrochloric acid (HCl)EQUIPMENT. Syringes (1 ml; Becton Dickinson, cat. no. 309602). 271/2 G, 1/2 inch needle for injections (Becton Dickinson, cat. no. 305109). Perfusion pump (e.g., Cole-Parmer Instrument, cat. no. 77200-60). Glass vials or centrifuge tube (15 ml, 17  120 mm2, Sterile, PPN; Greiner
Bio-One, cat. no. 188261). Cryostat (e.g., Leica CM1850; Leica). 24-Well tissue cell culture polystyrene plates (e.g., Sarstedt, cat. no. 83.1836). Micro cover glasses (24  60 mm2) (VWR, cat. no. 48404 454)
. Epifluorescent microscope (e.g., Nikon Eclipse 80i; Nikon ACT-1; Nikon). Confocal microscope (e.g., Olympus IX81DUS, Olympus; Image-Pro AMS)
. Analysis software (e.g., Image J; NIH) or other commercially available
analysis software. Water maze pool (see EQUIPMENT SETUP). Water maze tracking software (e.g., WaterMaze, Actimetrics)
REAGENT SETUP
Mice In our experiments we use offspring from a cross between C57BL/6
NTacfBr [C57B6] and 129Svev [129] mice (Taconic). We breed these mice in ourcolony at The Hospital for Sick Children. This hybrid strain is particularly suitedfor these types of experiments, since they learn well in the water maze andexhibit long-lasting water maze memories17,28,78. Other strains of mice may be
used (e.g., C57B6 mice), but the amount of training and durability of memory
may differ between strains31,78. Mice should be maintained on a 12-h light/darkcycle with free access to food and water. Mice should be at least 8 weeks of age

at the start of experiments, and behavioral procedures conducted at the sametime each day. Experiments should be conducted blind to the treatment
condition of the mouse. ! CAUTION Several factors including housing
conditions and age influence adult neurogenesis, and so care should betaken to match these variables across groups. ! CAUTION All experiments
must be performed in accordance with relevant authorities’ guidelines andregulations.
PBS, 0.1 M, pH 7.4 In 1–2-l beaker, add 2.7 g of NaH2PO4, 11.5 g of Na2HPO4
and 9 g NaCl. Add 1,000 ml of distilled water and stir. Measure pH, whichshould be B7.4.
BrdU solution Heat PBS solution to 40–50 1C, and slowly dissolve BrdU
by vortexing. Allow the BrdU injection solution to cool to room temperature(18–22 1C), and use immediately or store at 4 1C and use within 3 d.
Longer-term storage is not recommended due to the formation of white
BrdU precipitates (crystals). ! CAUTION There is some evidence that BrdU
may be carcinogenic and produce developmental abnormalities when given
to prenatal and neonatal animals1,15. The compound should be handled in
the fume hood. BrdU should be handled with gloves.
Blocking solution, 0.1 M PBS, 0.3% Triton X-100, 2% serum In a 1–2-lbeaker add 20 ml of normal goat serum, 3 ml of Triton X-100, 0.1 M PBS up to
1,000 ml and stir. Store in 50-ml aliquots at 20 1C.
4% Paraformaldehyde in 0.1 M PBS In 1–2-l beaker, heat B800 ml of 0.1 M
PBS to 60–65 1C while stirring. Once at 60–65 1C, add 40 g of paraformaldehyde
powder slowly while stirring. (Note: Adding a few drops of 1 N NaOH helps tokeep the solution clear.) Continue to stir until paraformaldehyde powder isdissolved, making sure that the temperature is maintained at 60–65 1C.
Let the solution cool to room temperature. Filter solution and store at
4 1C. ! CAUTION Prepare in the fumehood and handle with gloves.
May be stored at 4 1C for up to 1 week before using.
30% Sucrose in 0.1 M PBS In 1–2-1 beaker add B800 ml of 0.1 M PBS.
Weigh 300 g of sucrose and stir until fully dissolved. Fill the beaker to
1,000 ml with 0.1 M PBS.
Antibody dilution All antibodies are diluted in blocking solution.
EQUIPMENT SETUP
The water maze Large circular tanks are typically used as water mazes.
The absolute dimensions of water mazes vary greatly across studies, and there isno single correct size. However, water mazes designed for use with mice are
typically smaller (e.g., 100–150 cm in diameter) than those designed for use
with rats (e.g., 120–200 cm in diameter). The pool that we have used is 120 cm
in diameter and 50 cm deep. The pool is filled to a depth of 40 cm with water
made opaque by adding white nontoxic paint. Water temperature is maintained
at 28 ± 1 1C by a heating pad located beneath the pool. The escape or goal
platform may be circular or square. Our platform is circular and 10 cm indiameter, and represents B1/144th (or o1%) of the total pool surface. Our
platform is submerged 0.5 cm below the water surface and placed in one of thequadrants. The maze should be located in a sufficiently large room so that there
is at least 1 m between the perimeter of the pool and the surrounding walls.
Distinct, visual cues should be placed on the walls where they are clearly visible
to the mice. They must remain in a fixed position throughout the experiment.
There are a large number of commercially available tracking systems, which
output a large range of measures during both training and probe tests (e.g.,
WaterMaze (Actimetrics), HVS Image, Noldus). During training, latency toreach the platform is the most widely reported measure of performance(although path length is also often used). During the probe test, one or moreof the following are usually reported: time spent in each quadrant (percent
time or percent swim path), time spent in target zone versus other equivalent
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zones in pool (percent time), platform crossings (number) or average proximity(distance) are usually reported. Platform crossings are not recommended since
their relatively low frequency of occurrence increases variability, thereby
decreasing their sensitivity24. ! CAUTION Some laboratories maintain water ata lower temperature (e.g., B20 1C). However, lower temperatures may induce
significant hypothermia in mice, especially if short intertrial intervals are usedduring training79. The disadvantage of higher temperatures is that they may
encourage floating behavior. However, floating can usually be avoided byhandling mice extensively before beginning the experiment. ! CAUTION The
relationship between platform size and pool size will determine the difficulty ofthe task. Using a smaller platform or a larger pool will increase task difficulty,
and mice may not learn due to the lack of opportunities for reinforcement.
! CAUTION When platforms are submerged at depths 40.5 cm, there is anincreased tendency for mice to continue swimming even after finding theplatform. This is likely because the submerged platform does not offer sufficientescape from the water and is therefore less reinforcing. m CRITICAL Pools
that are too small (e.g., o1 m in diameter for mice), contain proximal
cues (e.g., markings on the inner wall of the tank) or distal cues that are
too close to the pool perimeter, may render the task hippocampusindependent.

PROCEDURE
Housing and acclimation
1| If mice are imported from a commercial vendor or other external source, allow mice to acclimate to the vivarium for atleast 1 week before beginning experiments.
! CAUTION Stress associated with travel may influence both adult neurogenesis51 and learning80, and therefore increase
variability. Mice should be group housed with littermates (three to five per cage), since social isolation negatively regulates
adult neurogenesis81 and learning82.
BrdU injections
2| Inject mice with BrdU (100–200 mg kg1; i.p.), two times per day for 5 d. In rodents, including mice, the S phase of thecell cycle lasts B8 h (ref. 21,83). Therefore, injections should be spaced at least 8 h apart in order to label distinct populationsof dividing cells. For an i.p. injection, the lower abdominal cavity should be isolated. In mice we use a 271/2-gauge needle anda 1-ml syringe.
! CAUTION Care should be taken not to stress the mice during the injection, since stress negatively regulates adult neurogenesis.
m CRITICAL STEP The goal of the BrdU administration is to label a subset of dividing cells in the dentate gyrus, rather than all
dividing cells. However, it is important that a sufficiently large sample is labeled, and so multiple injections over days are necessary
to label many cells undergoing mitosis.
’ PAUSE POINT The delay between BrdU injections and handling/water maze training depends on the experimental design(Fig. 4). Preferably, both short (e.g., 1 week) and long (e.g., 6 weeks) delays should be used. For example, in our study wefound minimal incorporation of newborn neurons using short BrdU injection-training delays and maximal incorporation of
newborn neurons when delays of 6 weeks or longer were used17.
Handling and water maze training
3| Before the commencement of training, individually handle mice for 2 min d1 for 7 d. Place mice in the palm of the hand,occasionally picking up and replacing into hand to mimic handling during water maze training procedures.
m CRITICAL STEP Handling is important since it allows mice to get used to experimenter, and minimizes potentially confounding
effects of stress and anxiety on learning and adult neurogenesis (e.g., survival).
4| Before training, mark the tails of mice with an indelible marker (or some other easy-to-see identifier). Since mice are grouphoused, tail-marking before the experiment facilitates identification during the behavioral procedures, thereby reducing delays
between mice and minimizing disturbances to other littermates.
5| To start a block of training trials, place mouse on the platform in the pool for 15 s. Once 15 s has elapsed, pick up themouse and place in pool at one of the four start positions (e.g., N, S, E or W). When placing the mouse in the pool, release themouse gently with the mouse facing the wall. Start timer.
m CRITICAL STEP In order to minimize stress associated with handling, when possible avoid picking the mouse up by its tail.
For example, when picking the mouse up from platform, slide your hand under the mouse and allow the mouse to climb into thepalm of your hand.
6| Finish the trial when the mouse finds the platform or 60 s have elapsed (whichever occurs first). During the first few trials,
the majority of mice will fail to find the platform. When this happens, at the end of the trial guide the mouse to the platform(e.g., because mice have a tendency to swim toward the experimenter, placing your finger on center of platform is sufficient toguide the mouse toward it).
7| Initiate a new trial using a new start position and repeat Steps 5–6. There are many possible training protocols for micein the water maze. Because the goal of this training is to produce a robust and long-lasting spatial memory, we recommend a
relatively intensive training schedule (six trials per day, presented in two blocks of three trials with an interblock interval ofB1 h and an intertrial interval of B15 s). The order of start locations should be pseudo-randomly varied throughout training.
Using these procedures, C57B6/129 mice learn rapidly and are able to express a robust spatial memory for a remarkably long
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time, with little decrement in performance even with retention delays of 9 weeks17. Even 3 d of training is sufficient to produce
a memory lasting at least a month in C57B6/129 mice28. However, more prolonged or more intensive training may be necessary
to produce enduring memories in other mouse strains such as C57B6 (ref. 28).
m CRITICAL STEP Spaced (e.g., two blocks of three trials), rather than massed (six trials in a row), presentation of training trials is
more effective at producing stable long-term spatial memory82,84, and so is recommended here.
? TROUBLESHOOTING
Water maze testing8| Either days or weeks after the completion of training (delay depends on experiment) give mice a series of probe tests toassess their spatial memory. In the probe test, place mouse on the platform for 15 s, and then pick the mouse up from theplatform, remove the platform from the pool, and place mouse in the pool in a unique start position. (We normally use a start
position 1801 opposite the platform location). Finish probe test once 60 s has elapsed by removing mouse from pool. For eachmouse, repeat two more times, with an intertrial interval of B3 min. Several measures of performance may be used to assess
spatial memory. These include the time spent in quadrant (percent time or percent swim path), time spent in target zone versus
equivalent zones in pool (percent time), platform crossings (number) or average proximity to site of platform (distance).
! CAUTION In our experiments, we give mice three probe tests to maximize the induction of Fos in dentate granule cells
associated with memory recall. However, one potential disadvantage of using multiple probe trials is that this may lead to theextinction of spatial memory. Since extinction involves the formation of a new inhibitory memory that competes with theoriginal memory for control of behavior85, it might be difficult to distinguish the Fos expression associated with the reactivationof the original spatial memory from the Fos expression associated with formation of a new extinction memory. However, wehave found that three probe tests produce minimal extinction (although this may vary in different mouse strains).
m CRITICAL STEP Before and after the probe tests, mice should remain minimally stimulated to avoid nonspecific induction of
Fos. In addition, care should be taken to ensure that procedures (e.g., transport of mice, housing and handling of mice, lighting
conditions) are, as much as possible, identical to those used during training to avoid Fos induction associated with novelty.
Anesthesia
9| Ninety minutes after the start of the probe tests, anesthetize mice with chloral hydrate (400 mg kg1) or other approved
anesthetic. After B1–2 min, check if mouse is fully anesthetized. A fully anesthetized mouse should display the following
properties: respiratory (breathing) rate should be regular and relaxed; withdrawal reflexes should be absent (e.g., lightly pinchtail); responses to external stimuli should be absent (e.g., no response to blowing air on the eye). If mouse is not fully
anesthetized, inject mice with 25% of original dose and repeat monitoring.
! CAUTION To prevent blood clotting, the heart should still be beating at the time of the perfusion. Therefore, care should betaken not to overdose the mouse.
Transcardial perfusion
10| Expose heart using dissecting tools.
11| Insert needle connected to pump into the left ventricle. Make an incision in the right atrium to allow blood to flow out of
the mouse’s body. Needle may be held in place with small clamps.
12| Perfuse the mouse with 0.1 M PBS (rate 10–12 ml min1).
13| Once the draining blood becomes clear, perfuse the mouse with 4% (wt/vol) paraformaldehyde in 0.1 M PBS(rate 10–12 ml min1).
! CAUTION When switching from PBS to 4% paraformaldehyde avoid air bubbles, since this may result in blood coagulation.
m CRITICAL STEP Poor-quality perfusions will likely lead to poor immunohistochemical results. For example, incomplete clearanceof blood is associated with autofluoresence of tissue, and poor fixation will lead to degradation of antigens. There are several hallmarksof a good perfusion, including the rapid, jerky movement of the limbs, whitening of the liver and paws and stiffening of the body.
Dissection
14| Using appropriate dissecting tools, remove the head and then remove the muscle and membranous tissue from the top partof the skull and gently extract the brain from the skull.
15| Cut the trigeminal and optic nerves, and let the brain fall into a beaker of cold 4% paraformaldehyde in 0.1 M PBS.
Postfixation
16| Immerse brain in the 4% paraformaldehyde in 0.1 M PBS for 24–48 h at 4 1C.
m CRITICAL STEP Overfixation (42 d) may result in the lack of staining due to unavailability of antigens.
’ PAUSE POINT Brain tissue can be left for 1–2 d in the 4% paraformaldehyde in 0.1 M phosphate buffer at 4 1C.
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17| Remove brain from the 4% paraformaldehyde in 0.1 M PBS solution, and immerse the brain in 30% sucrose in 0.1 M PBS
solution until the brain sinks to the bottom (usually takes 24–48 h).
’ PAUSE POINT Brain tissue can be left for 1–2 d in the 30% sucrose in 0.1 M PBS at 4 1C.
? TROUBLESHOOTING
Sectioning
18| Section tissue into 50-mm coronal slices using a cryostat. Transfer sections to 24-well plates loaded with 0.1 M PBS(pH 7.4), including 0.02% sodium azide. Collect four sets of sections per brain. Typically, we collect 40–48 sections per dentate
gyrus, and so there are 10–12 sections per set.
m CRITICAL STEP Sections should be cut immediately following Step 17. Brain tissue kept in 30% sucrose in 0.1 M PBS solution for
41 week might affect the morphology and compromise analysis.
’ PAUSE POINT Sections may be stored at 4 1C for several weeks.
DNA denaturation
19| Rinse sections three times, 5 min each with 0.1 M PBS (pH 7.4) on a shaker.
20| Denature DNA by incubating sections in 1 N HCl for 30 min at 45 1C.
m CRITICAL STEP Denaturating the cellular DNA into single strands is necessary to expose incorporated BrdU to the BrdU antibody.
Various denaturation procedures (ethanol treatment, enzyme treatment) may be used45. However, we recommend using theheated HCl treatment since it results in more effective exposure of the halogenated-nucleotide antigen. This step is critical,
and careful adjustments may be necessary to optimize results. Incomplete denaturation makes it difficult to detect BrdU-labeledcells, while some harsher denaturation procedures may damage tissue. For example, incubation with high concentrations of
HCl (42 N HCl) at high temperatures (465 1C) can be detrimental to other antigens (particularly surface antigens and receptors),
and so will interfere with Fos immunohistochemistry.
21| Neutralize the acid by rinsing sections three times, 5 min each with 0.1 M PBS (pH 7.4) on a shaker.
? TROUBLESHOOTINGBrdU and Fos immunohistochemistry22| Incubate sections with BrdU (1:500–1:1,000) and Fos (1:5,000–1:10,000) primary antibodies diluted in blocking solution
for 48–72 h at 4 1C on a shaker. To control for nonspecific BrdU staining, BrdU immunohistochemistry may be conducted incontrol mice that were not injected with BrdU. To check for nonspecific cross-reactions, omit the addition of the primary antibody.
’ PAUSE POINT Sections can be left for 2–3 d at 4 1C.
23| Rinse sections three times, 5 min each in 0.1 M PBS (pH 7.4) on a shaker.
24| Incubate sections with fluorochrome-conjugated secondary antibodies (Alexa Fluor 488 chicken anti-Rat and Alexa Fluor
568 goat anti-Rabbit; 1:500 to 1:1,000) in the dark, diluted in 0.1 M PBS (pH 7.4) with 0.3% Triton-X solution for 2 h at room
temperature on a shaker. Detergents such as Triton-X can be added to the secondary antibody diluent to reduce hydrophobic
interaction between tissue and reagent proteins, thus reducing nonspecific binding of secondary antibodies.
! CAUTION Exposing tissue to light can lead to photobleaching. Wrap 24-well plates in aluminum foil to avoid exposure to light.
25| Rinse sections three times, 5 min each in 0.1 M PBS (pH 7.4) on a shaker.
26| Carefully transfer sections to slides using a soft brush.
27| Add antifade mounting medium (PermaFluor) and place coverslips. Allow to dry for 2 d.
’ PAUSE POINT Mounted brain sections at 4 1C may be stored in the dark for up to 2 months before imaging with microscope.
Quantification
28| Using epifluorescent and confocal microscopes, quantify numbers of BrdU+ (option A), Fos+ (option B) and BrdU+/Fos+ (optionC) cells. Quantification may be limited to the granule cell layer17 or additionally include cells in the subgranular zone (defined as a
two-cell-body-wide zone bordering the granule cell layer and the hilus) (Fig. 5a). Because Fos expression in the subgranular zone is
generally lower, quantification of BrdU-labeled cells in this region will lead to a modest reduction in the proportion of BrdU-labeledcells expressing Fos. We do not recommend quantifying BrdU-labeled cells in the hilus as the contribution of hilar cells to learningand memory is poorly understood. Numbers of Fos+, BrdU+ and Fos+/BrdU+ cells may be normalized per fixed area of the dentate
gyrus or per section. Alternatively, absolute numbers for the entire dentate gyrus may be projected86.
m CRITICAL STEP Quantification should be conducted blind to experimental condition. Where possible, counts should be verified
by a second observer.
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PROTOCOL(A) Fos
Figure 5 | Examples ofa(i) Using an epifluorescent microscope Fos- and 5-bromo-2¢deoxyuridine (BrdU)with a 10 objective, take 2Dlabeling in the dentate
images of sections containing the
gyrus. (a) Low
dentate gyrus.
magnification merged(ii) Using threshold-based software
image of the dentate
gyrus showing Fos(e.g., Image J), quantify Fos+
immunofluorescence
cells in these sections. Gradually,(red), BrdUbcd
increase the threshold so that all
immunofluorescence+
Fos cells above background are(green) NeuN
immunofluorescenceselected.
(iii) Record total area of dentate gyrus (blue). Arrow heads
mark three BrdU-labeledfor each section.
Fos
BrdU
Mergedefgcells located within the! CAUTION Sections are relatively granule cell layer.
thick (50 mm). Therefore, there isWithin the granule cell
layer, note that thea possibility that exposure to themajority of BrdUFos antibody may not be uniform
labeled cells are locatedthroughout the section, resulting
in the innermost third. In contrast, the majority of Fos-labeled cells are located in the outmost regions ofin fainter labeling of the cells
the granule cell layer. (b–d) Examples of BrdU-labeled cells in the dentate gyrus. BrdU-labeling may belocated in the middle of the(b) uniform, or more patchy (c,d), possibly due to dilution of BrdU signal through redivision. Note that
section. The threshold should bedespite the patchy labeling, it is evident that the cell is appropriately shaped (round) and sized (B10mm). Representative confocal images of (e) Fos+ (red), (f) BrdU+ (green) and (g) Fos+/BrdU+ (arrow) cells
carefully adjusted to include
in the dentate gyrus after probe testing (scale bar ¼ 20 mm). Adapted from Kee et al.17.
faintly labeled cells that areclearly distinguishable frombackground noise. Fos-labeled cells should be appropriately sized (B10 mm in diameter) and shaped (round).
? TROUBLESHOOTING(B) BrdU(i) Using an epifluorescent microscope with a 40 objective, quantify BrdU+ cells manually, moving in and out of focus
throughout the entire granule cell layer. Scrutinize putative BrdU+ cells for size of nucleus (B10 mm in diameter) andshape (round). Note that for some BrdU+ cells, labeling of nucleus may be nonuniform and appear patchy (Fig. 5b–d).
! CAUTION BrdU-labeled cells may redivide and this raises two concerns. First, redivision may compromise the temporal
resolution of labeling. However, the vast majority of redivision is limited to a few days immediately after BrdU treatment,
and therefore temporal resolution is only minimally compromised35,39. Second, multiple rounds of redivision may lead todilution of the BrdU signal. While dilution of signal would be a problem in quantifying the absolute number of BrdU+/Fos+
cells, it will not affect quantification of the proportion of BrdU-labeled cells that express Fos (the primary dependentmeasure in these studies).
? TROUBLESHOOTING(ii) BrdU/Fos double-labeling. Using an epifluorescent microscope with a 40 objective, quantify the phenotype (Fos+/Fos–) of
all identified BrdU+ cells manually, moving in and out of focus throughout the entire granule cell layer.
(iii) Verify identified double-labeled cells on a confocal microscope with a 40 objective (Fig. 5e–g).
! CAUTION To exclude false double-labeling due to the overlay of signals from different cells located in the same z-axis,
analyze BrdU+ cells by moving through the entire z-axis of each cell to ensure signals come from the same source. To
avoid cross-detection of signals (e.g., Alexa 488 signals in the Alexa 568 channel or vise versa), the fluorescent images
should be examined sequentially for each channel.
! CAUTION We recommend quantifying Fos+, BrdU+ and BrdU+/Fos+ cells through the entire anterior–posterior extent of thedentate gyrus for two reasons. First, the low frequency of BrdU+/Fos+ cells renders sampling approaches (e.g., stereologicalcounting) less appropriate. Second, in the dentate gyrus Fos labeling is sparse after behavioral testing (typically o2.5 %of all cells). Therefore, overlap in the z-axis is rare, and permits 2D images to be used for quantification of Fos labeling.
? TROUBLESHOOTINGTIMING
Step 1, housing and acclimation: 1 week
Step 2, BrdU injections: 5 d
Step 3, Handling: 1 week
Steps 4–7, water maze training: 5 d
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PROTOCOL© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

Step 8, probe test and Steps 9–15, perfusion: 1 d
Steps 16 and 17, postfixation: 3–4 dStep 18, sectioning: 2–3 d
Steps 19–21, DNA denaturation: 2–3 d
Steps 22–27, BrdU and Fos immunohistochemistry: 1 d
Step 28, quantification: 1–4 weeks? TROUBLESHOOTING
Step 7, Morris water maze
Performance problems are somewhat more common in mice compared to rats. The most common of these are difficulty climbing
onto the platform, a propensity not to remain on the platform during the intertrial interval and floating. The frequency of these
performance problems varies greatly amongst strains, but extensive handling before the commencement of training is the best
approach to reduce the incidence of these problems. If they do occur, then the important decision is how to treat these mice.
The general rule is persistence. These problems occur more frequently on the first day of training, but, in most cases, will
disappear without any experimenter intervention over subsequent days. For example, mice that choose to swim during theintertrial interval (rather than stay on the platform) eventually get tired and learn to stay on the platform once they find it.
The one issue that may persist is floating. While some experimenter interventions effectively reduce floating (e.g., startling
the mouse by clapping hands), they are difficult to apply uniformly. Since persistent floating is relatively uncommon, mice thatpersistently float should be removed from the experiment.
More troubleshooting advice can be found in Table 1.
TABLE 1 | Troubleshooting table.
Step
16–17

Problem
Fos: Poor positive staining and/or
no positive staining with little orno background staining

Possible reason
Overfixation with 4% paraformaldehyde
in 0.1 M PBS

Solution
Do not leave brain tissue in fixative for 424 h

19–21

BrdU: Poor positive staining and/or
no positive staining with little orno background staining

Denaturation of DNA by hydrochloric acid(HCl) was not sufficient

Use higher concentration of HCl and/or
longer incubation time. Also use increased
temperature with HCl incubation

22–28

5-Bromo-2¢-deoxyuridine (BrdU)/Fos: BrdU/Fos primary and secondary antibody
concentration was not optimal
Poor positive staining and/or no
positive staining with little or nobackground staining
Incubation time with primary and/or
secondary antibody was too short

BrdU/Fos: Nonspecific and/or highbackground staining

3042 | VOL.2 NO.12 | 2007 | NATURE PROTOCOLS

Titrate BrdU/Fos primary antibody upon
arrival as concentrations may vary across batches

Use a longer incubation time for primary antibody
and/or secondary antibodies

The primary antibody does not recognize
the antigen due to incorrect fixation or
overfixation

Include antigen retrieval step. Note: For BrdU
primary antibody, overfixation does not affectBrdU immunostaining

Brain sections were left to dry

Do not let sections dry out and keep wet at all
times during the staining procedure

Nonspecific binding of primary or secondary Increase the number and time of washes inantibody
between steps
Incubation time with BrdU/Fos primary
antibody was too long

Reduce BrdU/Fos primary antibody
incubation time

Blocking reaction failed to prevent nonspecific binding of the secondary antibodies

Increase length and/or concentration of incubation with blocking solution made from the same
species as the host of the secondary antibody

Aggregates binding

Centrifuge antibody stock briefly in microcentrifuge at high speed to remove aggregates

Cross-reactivity between secondary
antibodies

Source of both secondary antibodies should
ideally come from same host (e.g., goat anti-rat
and goat anti-rabbit)

PROTOCOL© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

ANTICIPATED RESULTS
The outlined protocol utilizes immunohistochemical approaches to visualize the recruitment of new neurons into the circuits
supporting water maze memory in intact animals. BrdU treatment should result in robust labeling of new neurons in the dentate
gyrus. These should be predominantly located in the innermost layers of both the upper and lower blade of the dentate gyrus.
Furthermore, numbers of BrdU-labeled cells should decrease as the post-BrdU treatment survival times lengthen. After water
maze testing, induction of Fos should be expected in B0.5–2.5% of granule cells. Fos expression is typically more prevalent in
the outermost layers of both the upper and lower blade of the dentate gyrus. The overlap between these two populations should
be sensitive to the delay between BrdU treatment and training in the water maze. At delays beyond 4 weeks, maximal numbersof BrdU+/Fos+ cells are expected, indicating that as new neurons mature they become increasingly likely to be incorporated into
the circuits supporting spatial memory in the dentate gyrus.

ACKNOWLEDGMENTS We thank S. Josselyn and M. Sakaguchi for comments on thismanuscript. This work was supported by grants from Canadian Institutes of HealthResearch (CIHR), Natural Sciences and Engineering Research Council (NSERC) andthe EJLB Foundation (P.W.F.). N.K. and A.H.W. were supported by the Hospital for
Sick Children Restracomp awards. C.M.T. received support from the Graduate
Program in Areas of Basic and Applied Biology (GABBA) and the Portuguese
Foundation for Science and Technology (FCT).
Published online at http://www.natureprotocols.com
Reprints and permissions information is available online at http://npg.nature.com/
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