ABC monoclonal antibody staining of dissected larval and adult CNS
Ito, 20.1.97
For 1st-instar CNS, use PBS instead of PBT throughout the process (small specimen is too fragile for the detergent).
for larva / pupa : Dissect CNS in PBS
for adult :Put the animal one minute in 50-70% EtOH (to wash oil on the body surface)
Transfer to PBS and dissect the CNS
Fix:

Wash:
Block 1:Wash:
Block 2:
1st Ab:Retrieve:Wash:
2nd Ab:Retrieve:Wash:

put 100-200 µl fixative into Eppendorf tubes.
most cases: 4% Formaldehyde (FA) / PEM (100µl 37% FA / 900µl PEM, 400 µl 10% FA / 600µl PEM)
using “ EM grade ultra-pure FA” yields better results
Pool dissected tissues in the fixative for up to 10-20 min
Change to fresh fixative and incubate R.T. further 50 min (for 1st instar: 25 min)
2 times 5 min with PBT (0.3-0.5 % Triton X /PBS)
For keeping the specimen at 4°C (up to a few days), wash once more with PBT.
with 0.3% H2O2 / Methanol (100µl 30% H2O2 / 10 ml Methanol)
R.T. 30 min
to kill endogenous peroxidase
4 times 5 min with PBTwith 1.5% horse serum / PBT (150µl / 10 ml PBT)
R.T. >1 hr
to fill nonspecific antibody binding region
monoclonal antibody (mouse) (1:500 in 1.5% horse serum / PBT)
4°C Over Night or R.T. >3 hr
keep used antibody in 4°C (used antibodies works better; up to a few weeks or a few staining sessions)
3 times 5 min with PBTBiotin-conjugated horse anti-mouse IgG (Vectastain 1:500 in 1.5% horse serum / PBT)
R.T. >2 hr
keep used antibody in 4°C
3 times 5 min with PBS (to wash out Triton X before avidin-biotin reaction and DAB staining)ABC:

Mix 10µl Solution A, 10µl solution B, 500µl PBS (Vectastain Elite)
keep R.T. >30 min before usethen incubate the specimen R.T. >1 hr
Wash:
4 times with PBS

Stain:

0.5-1 mg/ml DAB + 0.03% H2O2 (1 µl 30% H2O2 / 1 ml DAB)
1.5% H 2O2
1.3µl
6.7µl

Wash:
Post Fix:
Wash:
Dehydrate:
Mount:

1mg/ml DAB
200µl1000µl
R.T. 5-20 min (check the staining under dissecting microscope)
2 times 5 min with PBS
2% Glutaraldehyde (GA) / PBS (80 µl 25% GA / 920 µl PBS)
R.T. > 1 hr
3 times 5 min with H2O

keep hazardous DAB waste !
keep hazardous DAB waste !
keep hazardous DAB waste !
keep hazardous DAB waste !

30%, 50%, 70%, 90% each 3 min -> 100% 5 min -> 100% 10 min -> Xylen 15 min -> Araldit 30 minchange Araldit again
in Araldit

Sigma DAB Tetrahydrochloride Tablet (D5905, 100 tablets)1

antibody / Fluorescent staining of dissected larval and adult CNS
Ito, 21.1.99
For 1st-instar CNS, use PBS instead of PBT throughout the process (small specimen is too fragile for detergent).
for Larva :for adult :Dissect CNS in PBS
Put the animal one minute in 50-70% EtOH (to wash oil on the body surface)
Transfer to PBS and dissect the CNS

Fix:

4% Formaldehyde (FA) / PEM (100µl 37% FA / 900µl PEM, 400 µl 10% FA / 600µl PEM)
R.T. 50 min (1st instar: 25 min) with gentle agitation

Wash:

4 times 5 min with PBTBlock:

with 10% goat serum / PBT
R.T. >1 hr
to fill nonspecific antibody binding region

1st Ab:antibody in 10% goat serum / PBT4°C Over Night or R.T. >3 hr
keep used antibody in 4°C (used antibodies works better; up to a few weeks or a few staining sessions)
3 times 5 min with PBTRetrieve:Wash:
2nd Ab:Cy3-conjugated anti IgG (Jackson, 1:100 in 10% goat serum / PBT)
(anti mouse IgG for monoclonal, anti rabbit IgG for most polyclonal)
R.T. >2 hrduring incubation, put cover onto the Eppendorf tubes to keep specimen in the dark (in all the steps)
Wash:
3 times 5 min with PBS
Dehydrate:
Mount:

in 50% Glycerol / PBS for 1 hour
in 50% antifade Glycerol / PBS for 30 min
in 50% antifade Glycerol / PBS

Store:

keep in refrigerator until observation (up to 1 week)

antifade

0.25 % n-propyl gallate 50% Glycerol, PBS
dissolve 25 mg n-propyl gallate in 5 ml PBS
add 5 ml Glycerol
adjust pH to 8.0-8.6 with 1 N NaOH (50-80 µl)

1st :Cappel Rabbit polyclonal anti ß-gal
Promega Mouse monoclonal anti ß-gal
Sigma Mouse monoclonal anti bovine-tau
Sigma Rabbit polyclonal anti-WGA

Cosmo Bio: 55693 (3712-0081)
Z 3781
T5530
T4144

2nd:

Jackson AffinitiPure Goat Anti-Mouse IgG (H+L) Cy3-conjugated115-165-146
Jackson AffinitiPure Goat Anti-Rabbit IgG (H+L) Cy3-conjugated115-165-144
Cappel: affinity purified goat anti rabbit IgG (H+L) - FITC conjugated (whole)
Cosmo Bio: 55662(1612-0081)
Zymed: affinity purified goat anti rabbit IgG (H+L) - FITC conjugated (whole)

Cosmo Bio: 62-6111

Preabsorbtion: If background is high, incubate the diluted antibody for >2 hrs with fixed embryos (dechorionate withbreach for 4 min, fix with heptan / 10% FA for 30 min , deviterinize with heptan/methanol, wash with methanol andthen with PBS)

2GFP preparation of dissected larval and adult CNS
Ito, 20.1.99
for Larva :for adult :Dissect CNS in PBS
Put the animal one minute in 50-70% EtOH (to wash oil on the body surface)
Transfer to PBS and dissect the CNS

put 100-200 µl 4% Formaldehyde (FA) / PEM into Eppendorf tubes.
(100µl 37% FA / 900µl PEM, or 400 µl 10% FA / 600µl PEM)
Pool dissected tissues in the fixative for up to 10-20 min
Change the fresh fixative and incubate R.T. further for 50 min with gentle agitation
during incubation, put cover onto the Eppendorf tubes to keep specimen in the dark (in all the steps)

Fix:

Wash:

3 times 5 min with PBS

Dehydrate:
Mount:

in 50% Glycerol / PBS for 1 hour
in 50% antifade Glycerol / PBS for 30 min
in 50% antifade Glycerol / PBS

Store:

keep in refrigerator until observation (up to 1 week)

antifade

0.25 % n-propyl gallate 50% Glycerol, PBS
dissolve 25 mg n-propyl gallate in 5 ml PBS
add 5 ml Glycerol
adjust pH to 8.0-8.6 with 1 N NaOH (50-80 µl)

3Anti ß-gal DAB staining of dissected larval and adult CNS
Ito, 4.6.95
For 1st-instar CNS, use PBS instead of PBT throughout the process (small specimen is too fragile for detergent).
for Larva :for adult :Dissect CNS in PBS
Put the animal one minute in 50-70% EtOH (to wash oil on the body surface)
Transfer to PBS and dissect the CNS

Fix:

put 100-200 µl fixative into Eppendorf tubes.
most cases: 4% Formaldehyde (FA) / PEM (100µl 37% FA / 900µl PEM, 400 µl 10% FA / 600µl PEM)
some cases: 1% Glutaraldehyde (GA) / PBS (40µl 25% GA / 960µl PBS)
GA works well with anti ß-gal, but some antibodies don’t work with GA at all.
Pool dissected tissues in the fixative up to 10-20 min
Change the fresh fixative and incubate R.T. further
FA: 50 min, FA for 1st instar: 25 min, GA: 10 min
2 times 5 min with PBT (0.3-0.5 % Triton X /PBS)
For keeping the specimen at 4°C (up to a few days), wash once more with PBT.

Wash:
Block 1:Wash:
Block 2:
1st Ab:Retrieve:Wash:
2nd Ab:Retrieve:
Wash:with 0.3% H2O2 / Methanol (1/100 volume of 30% H2O2 )R.T. 30 min
4 times 5 min with PBTwith 10% goat serum / PBT
R.T. >1 hr

for HRP: Peroxidase-conjugated anti rabbit IgG (Cappel, 1:500 in 10% goat serum / PBT)
for ABC: Biotin-conjugated anti rabbit IgG (Vectastain 1:500 in 10% goat serum / PBT)
R.T. >2 hr
keep used antibody in 4°C
3 times 5 min with PBS (to wash out Triton X before avidin-biotin reaction and DAB staining)
goto Stain
Mix 10µl Solution A, 10µl solution B, 500µl PBS (Vectastain Elite)
keep R.T. >30 min before usethen incubate the specimen R.T. >1 hr
Wash:
4 times with PBS

Stain:

0.5-1 mg/ml DAB + 0.03% H2O2 (1 µl 30% H2O2 / 1 ml DAB)
1.5% H 2O2
1.3µl
6.7µl

Post Fix:
Wash:
Dehydrate:
Mount:

to fill nonspecific antibody binding region

rabbit polyclonal anti ß-gal (Cappel 1:1000 in 10% goat serum / PBT)
4°C Over Night or R.T. >4 hr
keep used antibody in 4°C (used antibodies works better; up to a few weeks or a few staining sessions)
3 times 5 min with PBT

HRP:
ABC:

Wash:

to kill endogenous peroxidase

1mg/ml DAB
200µl1000µl
R.T. 5-20 min (check the staining under dissecting microscope)
once 5 min with PBS
2% Glutaraldehyde (GA) / PBS (80 µl 25% GA / 920 µl PBS)
R.T. > 1 hr
3 times 5 min with H2O

keep hazardous DAB waste !
keep hazardous DAB waste !
keep hazardous DAB waste !
keep hazardous DAB waste !

in 50% Glycerol / H2O for 1 hourin 80% Glycerol / H2O for 30 min
in 80% Glycerol / H2Oor:
For plastic mount:
Dehydrate: 30%, 50%, 70%, 90% each 3 min -> 100% 5 min -> 100% 10 min -> Xylen 15 min ->Mount:in Araldit
Stock
1st :
2nd

DAB tablet:
Goat Serum:

Cappel Rabbit polyclonal anti ß-gal
Cosmo Bio: 55693 (3712-0081)
affinity purified goat anti rabbit IgG (H+L) (whole)-- peroxidase conjugated : Cappel : Cosmo Bio: 55689 (3612-0081), Zymed : Cosmo Bio: 62-6120-- biotinilated (whole)
Cappel : Cosmo Bio: 55699 (8612-3711), Vector : Vectastain Elite
Sigma DAB Tetrahydrochloride Tablet
Sigma D-5905
CMN Chemicon
Cosmo Bio S-26 017

4Antibody staining of Embryos (with Vectastain kit)
ITO Kei, 1995 April
Dechorionate:
Wash:
Fix:

Deviterinyze:

Wash:collect eggs with a nylon mesh attached to the bottom of a funnel
soak the eggs in 7.5% Bleach in H2O (50% Chrolox, 50% H2O)
R.T. 4 minwith H2O
Transfer into an Eppendorf tube containing
FA / Heptan (1 ml)
400 µl PBS, 100µl 37 % Formaldehyde, 500 µl n-Heptan(or 100 µl PBS, 400 µl 10 % EM grade Formaldehyde, 500 µl n-Heptan)
R.T. 30-40 min
remove the FA+PBS phase (lower part)add the same amount of 100% Methanol
shake well by hand or by Vortex
remove the upper phase (Methanol)
add another volume of Methanol
shake well by hand or by Vortex
until the majority of the eggs go down to the bottom
remove heptan and Methanol solutions
2 times with Methanol
until eggs become non-sticky
Keep embryos at this stage at 4°C (up to a few weeks)

Before staining, wash 4 times with PBT (0.3-0.5% Triton X-100 in PBS)
Block:

with 10% goat serum / PBT
R.T. >1 hr
to fill nonspecific antibody binding region

1st Ab:monoclonal antibody (mouse) or polyclonal antibody (rat, rabbit, goat etc.)(in 10% goat serum / PBT) preabsorbed !
4°C Over Night or R.T. >3 hr
keep used antibody in 4°C (used antibodies works better; up to a few weeks or a few staining sessions)
3 times with PBTRetrieve:Wash:
2nd Ab:Retrieve:
Wash:
ABC:

Wash:
Stain:

Biotin-conjugated antibody (1:500 in 10% goat serum / PBT; Vectastain)
for monoclonal 1st: anti mouse Ig, for polyclonal 1st: anti rat, rabbit, goat,,, Ig
preabsorbed!
R.T. >2 hr
keep used antibody in 4°C
3 times with PBS (to wash out Triton X before avidin-biotin reaction)
Mix 10µl Solution A, 10µl solution B, 500µl PBS (Vectastain Elite)
keep R.T. >30 min before usethen incubate the specimen R.T. >1 hr
4 times with PBS
0.5-1mg/ml DAB + 0.01% H2O2
1.5% H 2O2

1.3µl

6.7µl

Wash:

1mg/ml DAB
200µl1000µl
R.T. 5-20 min (check the staining under dissecting microscope)
2 times with PBS

keep hazardous DAB waste !
keep hazardous DAB waste !

Wash:once with H2O

keep hazardous DAB waste !

Dehydrate:

in 50% Glycerol / H2O for 1 hourin 80% Glycerol / H2O for 30 min
in 80% Glycerol / H2O

Mount:

For plastic mount:
Dehydrate: 30%, 50%, 70%, 90% each 3 min -> 100% 5 min -> 100% 10 min -> Xylen 15 min ->Mount:in Araldit
Preabsorbtion: incubate the diluted antibody for >2 hrs with fixed embryos (dechorionate with breach for 4 min, fix withheptan / 10% FA for 30 min , deviterinize with heptan/methanol, wash with methanol and then with PBS)

5

X-gal staining of embryo
Ito, 4.6.95
Dechorionate:

put the eggs in 7.5% Bleach (in H2O)

Wash:

R.T. 4 mincollect the eggs with a nylon meshwith H2O
transfer to an Eppendorf tube

Fix:

GDA / Heptan (300-500 µl)
45 ml PBS, 5 ml 25% Glutardiardehid, 50 ml n-Heptan
store at 4°C, shake well just before use and take the upper phase
R.T. 15 min

Deviterinyze:(divitelinize by hand works better)remove GDA / PBS phase (lower part)add the same amount of 80% EtOH (in H2O)
shake well by hand or by Vortex(Although Ethanol deviterinization is not so effective as with
Methanol, the loss of ß-galactosidase activity is smaller.)remove the upper phase (EtOH)
add another 80% EtOH
shake well by hand
until the majority of the eggs go down to the bottom
remove heptan and EtOH solutions
several times with 80% EtOH
until eggs become non-sticky
wash 2 times with PBT (0.5% Triton X-100 in PBS)

Wash:

Stain:

heat X-gal buffer to 65°C
buffer:
100 ml
860 mg20.3 mg
109 mg
140 mg0.1 M Na phosphate buffer
NaCl
MgCl2
K3[Fe(III)CN6]
K4[Fe(II)CN6]

store at 4°C
add 1/100 volume of 20% X-gal
200 mg X-gal in 1 ml DMF, store at 4°C
mix well and gradually cool down to room temperature
If precipitation appears, heat and mix the solution again.
apply the staining solution to the eggs
37°C 2 hr to Over Night (with gentle shaking)Wash:

3 times 5 min with PBS

Dehydrate:

in 50% Glycerol / H2O for 1 hourin 80% Glycerol / H2O for 30 min
in 80% Glycerol / H2O

Mount:or:For plastic mount:
EtOH Dehydrate:
Mount:

70%, 90% each 5 min -> 100% 5 min -> 100% 10 min -> Xylen 15 minin Araldit6

X-gal staining of dissected larval and adult CNS
Ito, 4.6.95
for Larva :for adult :Dissect CNS in PBS
Put the animal one minute in 50-70% EtOH (to wash oil on the body surface)
Transfer to PBS and dissect the CNS

Fix:

put 100-200 µl fixative into Eppendorf tubes.
1% Glutaraldehyde (GA) / PBS(40µl 25% GA / 960µl PBS)
Pool dissected tissues in the fixative up to 10-20 min
Change the fresh fixative and incubate R.T. 15 min
2 times 5 min with PBT (0.3-0.5 % Triton X /PBS)

Wash:
Stain:

heat X-gal buffer to 65°C
buffer:
100 ml
860 mg20.3 mg
109 mg
140 mg0.1 M Na phosphate buffer
NaCl
MgCl2
K3[Fe(III)CN6]
K4[Fe(II)CN6]

store at 4°C
add 1/400 volume of 20% X-gal
200 mg X-gal in 1 ml DMF, store at 4°C
mix well and gradually cool down to room temperature
If precipitation appears, heat and mix the solution again.
apply the staining solution to the eggs
37°C 2 hr to Over Night (with gentle shaking)Wash:

3 times 5 min with PBS

Dehydrate:

in 50% Glycerol / H2O for 1 hourin 80% Glycerol / H2O for 30 min
in 80% Glycerol / H2O

Mount:or:For plastic mount:
EtOH Dehydrate:

70%, 90% each 5 min -> 100% 5 min -> 100% 10 min -> Xylen 15 min

Mount:in Araldit

7

Fixative
4 % Formaldehyde (FA)good for the first trial

10% Formalin / PBS
PBS Tablets pH7.4 (25 degree) (Sigma P4417)
4 % Ultra pure Formaldehyde (FA) usually the good and most convenient
40% EM grade Ultra pure Formaldehyde / PEM
Folmaldehyde, Methanol Free, 10%, Ultra pure EM grade (Polyscience, 04-1040-18)
4 % Paraformaldehyde (PFA)mix:

good for fixing protein

paraformaldehyde

4

g 1

g

0.1 M Phosphate Buffer

90

ml 22

ml

100

ml 25

ml

warm to 60°C (ca. 20 min.)cool and mess up to
store at 4°C (max 1 week )PFA for protein, periodate-lysine for sugar

PLP (Periodate–Lysine–Paraformaldehyde)
Stock A:mix:

Lysine–HCl
H2O 50add

0.1 M Na2HPO4 to pH 7.4

mess up with 0.1 M Phsphate Buffer to1.827 gml

0.365 g
10 ml

100

ml 20

8
ml

g 0.2 g5 ml

mlstore at 4°C (max 10 days )Stock B:mix:

Paraformaldehyde (PFA)H2O 100

warm to 60°C (ca. 20 min.)add 1N NaOH

a few drops to dissolve PFA

filtrate
store at 4°C (max only a few days )Just before use:mix

Stock A

7.5 ml9 ml

3 ml

Stock B
NaIO4

2.5 ml21.4 mg

3 ml
25.68 mg

1 ml
8.56 mg

12 ml
pH. 6.2 )

4 ml

total
10 ml
(final 0.01 M NaIO4 – 0.075 M Phosphate – 2 % PFA

for 1x soln.
10.22 g0.1 M Na-Phosphate Buffermix
Na2HPO4 . H2O
or
or
or
AND
or

Na2HPO4 . 2H2O
Na2HPO4 . 7H2O

12.82 g64.1 g19.30 g96.5 gNa2HPO4 . 10H2O
NaH2PO4 . H2O

25.79 g128.95 g

3.86 g19.3 g

NaH2PO4 . 2H2O

4.37 g21.85 gtotal

1l

500 mlfor 100 ml

for 500 ml

0.1 M PIPES (302.37)

3.0237 g

15.1185 g

2 mM EGTA (380.35)
1 mM MgSO4 . 7H2O (246.48)

76.07 mg24.648 mg

380.35 mg
123.24 mg

PEM Buffermix

for 10x soln.
51.1 gadjust pH to 6.95 with NaOH (or HCl)

8

Preparation of Araldit
Araldit CY 212(Serva, #13824, 1 kg)

Araldit hardener HY 964(Serva, #13826, 1 kg)

Araldit softener (Phtalic Acid Dibuthyl Ester: PADE)(Serva, #32805, 250 ml)

Araldit Beschleuniger DY 964(Serva, #13825, 100 g)

For semi-thin sectioning
100 ml Araldit CY 212 into a 500 ml glass cup.
Add 100 ml Araldit hardener HY 964 slowly.
Put the rotor of a low-speed electric mixer in it.
Let the mixer slowly rotate for at least 2 hrs. (avoid making bubbles in the solution.)
Add 10 ml Softener PADE very slowly (drop by drop) with the mixer rotating.
Adding as little as 500 ul at once will result in uneven solution,
which is not usable any more.
Mix for another 15 min.
Add 5 ml Beschleuniger DY 964 very slowly (drop by drop).
Mix for another 30 min.
Aliquot the Araldit in 10-20 ml disposable syringe.
Store frozen at -20°C.
For whole-mount preparation
Softener PADE is not needed. Mix CY 212, HY 964, and DY 964 in this order.
Araldit for semi-thin sectioning is also applicable to whole-mount preparation, although it costs a bit more.
***
Araldit monomer is harmful. Use gloves while handling. Wipe out with acetone in case of an accident.
Beschleuniger is a highly volatile material; work in a draft chamber.

After use, wipe the glass cup and mixing rotor with acetone and keep them at 63°C over night until
remaining araldit become polymerized.

9

Making Semi-thin Sections
Ito, 4.6.95
Larva, Pupa, Imago:
Dissect animals in Ringer or PBS
Embryo:Prefix with 25% GDA / Heptan
R.T. 10 min
Wash 3 times with PBS (Remove Heptan)
Fixation 1:
Fix with 6% GDA (Glutardialdehyd)/PBS
4°C, 1-4 hr
Wash 2 times with PBS
Fixation 2:
Fix with 2% OsO4/PBS
4°C, in the Dark, Over Night
CAUTION: AVOID SKIN and GAS CONTACT
Wash 3 times with PBS
Dehydrate and Embed:
30%, 50%, 70%, 90% each 3 min -> 100% 5 min -> 100% 10 min ->Xylen 10 min 2 times ->Araldit/Xylen (1:1) 6hr-Over Night in the Abzug (let Xylen evaporate) ->Araldit 8-12 hr (let residual Xylen evaporate) ->Araldit in the moldPolymerize 45°C, 12 hr -> 65°C, 36-48 hr
SectionStaining
Richardson's Solution
1% Toluidine blue and 1% azur II - 1% Borax
60°C, 15-60 sec.
or

TB Solution
1 ml 0.2% Toluidine blue / water
1 ml 1% Methylen blue / 1% Borax
18 ml water
60°C, 30 sec. - 2 min
Wash with running tap water
Wash with distilled water
Dry
Mount with Araldit

10Anti ß-gal staining of larval CNS original in Mainz
Dissect CNS
Fix:
1% Glutardialdehyde (GDA) / PBS (40 µl 25% GDA / 960µl PBS)
R.T. 10 min
GDA does not work with other antibodies! Anti ß-gal works with Formaldehyde.
Use Formaldehyde (FA) when staining embryos.
Wash:
2 times with PBS
Block 1:Wash:
Block 2:

with 0.3% H2O2 / Methanol
R.T. 30 min
to kill endogenous peroxidase
3 times with PBS
with 10% lamb serum / PBS
R.T. >1 hr
to fill nonspecific antibody binding region

1st Ab:anti ß-gal (rabbit polyclonal) (1:7000 in 10% lamb serum / PBS)
1:14 preabsorbed antibody solution (1:500)
4°C Over Night
Retrieve: keep used antibody in 4°C
Wash:
3 times with PBS
2nd Ab:

Biotin conjugated anti rabbit IgG (1:500 in 10% lamb serum / PBS)
(Vector stain)
preabsorbed!
R.T. 2 hr
Retrieve: keep used antibody in 4°C
Wash:
3 times with PBS
ABC:

Wash:
Stain:Wash:

Mix 10µl Solution A, 10µl solution B, 500µl PBS
keep R.T. >30 min before usethen R.T. 1 hr with the specimen
4 times with PBS
1mg/ml DAB + 0.01% H2O2
1.5% H2O2
1mg/ml DAB
200µl
R.T. 5-10 min
2 times with PBS

1.3µl
1000µl(40 µl 25% GDA / 960µl PBS)

Post Fix: 1% Glutardialdehyde (GDA) / PBS
R.T. 30 min
Wash:once with Water
Dehydrate:

6.7µl

30%, 50%, 70%, 90% each 3 min -> 100% 5 min-> 100% 10 min -> Xylen 15 min -> 50% Araldit / Xylen O/N-> Araldit 6 hr -> Araldit

11