TITLE: Chromogenic in situ hybridisation
AUTHORS: Stephen Carter

[DESCRIPTION]
The protocol for performing chromogenic in situ hybridisations in zebrafish embryos and larvae in the Wilson lab. 

[STEPS]
SECTION: Probe synthesis
2. If the template DNA is a plasmid, it must first be linearised by restriction enzyme digestion. If it is a PCR product, it can be used directly. 

Prepare probe synthesis reaction mix

Template DNA (PCR product or plasmid) -0.5-1 µg
10x DIG RNA mix (Roche) - 2 µL
10x transcription buffer - 2 µL
RNA polymerase (T3/T7/SP6) - 2 µL
100mM DTT - 2 µL 
RNase inhibitor - 0.5 µL
dH20 to final volume 20 µL

Mix and incubate for 120 min at 37-40 °C (temperature depending on the polymerase you are using-see polymerase sheet). Longer incubations, up to overnight, can be performed if yield is low with 2 hrs incubation. 
SECTION: Probe synthesis
3. OPTIONAL: add 2 µL of DNase1 to remove the template DNA; 15 min at 37 °C. We tend to skip this step. If you are doing a precipitation step it's better if you have the DNA template because it helps the precipitation of your RNA. The DNA is not DIG labelled so it does not affect your in situ reaction.
SECTION: Probe synthesis
4. Probe purification 

The best approach here is to perform a double purification of the probe, which removes almost all unicorporated nucleotides. This can allow you to use the probe at higher concentrations from the start without high background staining. 

First, perform a purification with Sephadex G-50 columns, such as ProbeQuant columns. If unavailable, use another RNA/probe purification kit. Follow the manufacturers instructions. 
 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             
SECTION: Sample collection
8. Perform in Eppendorf tubes or well plates, depending on number of samples. 

Fix embryos/larvae at desired stage in 4% PFA in PBS for 240 min at room temperature or 20 min at 4 °C.

Wash x4 with 1x PBS, 5 min per wash. 

 

SECTION: In situ: Day 3 (Development)
16. Wash x3 with PBST.

Wash multiple times in fresh AP buffer over 1-2 hrs. 

Prepare NBT/BCIP (Roche) solution (1 µL NBT, 3.5 µL BCIP, 1 mL AP buffer) just prior to use. 

Develop in situs in NBT/BCIP solution at room temperature. Protect samples from light at this point (e.g. cover with aluminium foil). Check development under a microscope every 5-10 minutes initially. 
It is easier to do this if the samples are in a plate or dish. Depending on the probe/target mRNA, development can take minutes to hours (even overnight/several days). 

 

Stop reaction with two quick PBST washes.

Post-fix with 4% PFA for 20 min at room temperature.

Wash x3 with PBS.

Transfer samples to glycerol through a graded series of glycerol/PBS washes (25%, 50% and 75%). Store samples in 100% glycerol at 4 °C, in the dark.

Image samples under a compound microscope or stereoscope, mounted in either glycerol or low-melting agarose in 80% glycerol. 
SECTION: Sample collection
9. Wash samples into methanol. Start with 50% for 10 min at room temperature, then 100%. 

Store samples 10 min at -20°C in methanol. 

Embryos or larvae can be stored in methanol at -20°C for several months. 
SECTION: In situ: Day 1 (Probe hybridisation)
10. Rehydrate samples: 

75% methanol/PBST 5 min
50% methanol/PBST 5 min
25% methanol/PBST 5 min
PBST 5 min (x4)

 

SECTION: In situ: Day 1 (Probe hybridisation)
11. Permeabilize samples with proteinase K digestion at room temperature. Dilute proteinase K stock (10mg/ml, 1000x) in PBST. Concentration and duration are stage dependent.

3-9 somites - 1x for 30 s-1 min
10-18 somites - 1x for 2 mins
24 hrs - 1x for 10 mins
2-3 dpf - 2x for 20-30 mins
4-6 dpf - 3x for 45mins - 1 hr
Larval brains - 1x for 1 min (or not at all)
Juvenile brains - 2x for 20 mins

Quickly wash twice with PBST (no incubation) to stop digestion.
SECTION: In situ: Day 1 (Probe hybridisation)
12. Post-fix with 4% PFA for 20 min at room temperature. 

Wash x4 with PBST for 5 min each.
SECTION: In situ: Day 1 (Probe hybridisation)
13. Pre-hybridize samples by incubation in hybridization buffer for 120 min at 68 °C. 

Remove hybridization buffer and add probe diluted in the same buffer. 

Incubate 120 min at 68 °C. 
SECTION: In situ: Day 2 (Antibody binding)
14. Remove probe and save for reuse (working probe dilutions can be used many times). 

Wash once with 2x SSC for 30 min (perform at 68 °C).


Wash twice with 0.2x SSC for 30 min each (perform at 68 °C).

 

SECTION: In situ: Day 2 (Antibody binding)
15. Wash with PBST for 1 hr with several changes.

 

Block in MaBl at room temperature for at least 120 min 

Incubate samples in anti-DIG-AP Fab fragments (Roche) diluted 1:6000 in MaBl 120 min at 4 °C. Leave samples on a plate rocker if possible. 

SECTION: Probe design
1. This in situ uses Digoxigenin-labelled antisense RNA probes. Probes are transcribed from DNA templates, which are themselves generally amplified by RT-PCR of mRNA extracted from zebrafish embryos, larvae or tissues. Antisense transcription is achieved by the addition of a promoter sequence to the reverse primer used in the RT-PCR. Add the following sequence to the beginning (5' end) of your reverse primer, depending on the polymerase being used:

T7 - GGATCCTAATACGACTCACTATAG
T3 - GGATCCATTAACCCTCACTAAAGG
SP6 - TATTTAGGTGACACTATAG

Generally speaking, T7 is the best of the three polymerases. 

Alternatively, the PCR product can be cloned into a expression vector (e.g. a TOPO vector) such that one of these promoters is at the 3' end of the template. This allows for long term storage and re-use of the probe, as the plasmid can be amplified by bacterial transformation. 

Probe length can vary from <200 to >1000 nucleotides. Somewhere in the middle of this range is usually considered ideal. The longer the probe, the more difficult it is for it to penetrate the sample. Shorter probes meanwhile are more prone to off-target binding, especially to similar mRNAs (e.g. paralogues).
SECTION: Probe synthesis
7. Store probe

Store the probe at -20 °C diluted at least 1:1 in hybridization buffer initially (the formamide protects the probe from degradation). The working concentration of a given probe is variable and requires optimization. Generally, 1:100, 1:250 or 1:500 are good starting points. 
SECTION: Probe synthesis
6. We find it best practice to at this point, check probe yield and integrity. Measure concentration using a Nanodrop; there should be no less than 100ng/uL. Run on a 1% agarose gel, ideally there should be crisp bands (smearing on a gel indicates RNA degradation)
SECTION: Probe synthesis
5. Next, perform ethanol precipitation on the elute:
Add 1/10 volume of 3M sodium acetate (pH 5) and 3 volumes of ice-cold 96-99% ethanol and mix well. 
Incubate at -20 °C  for at least  60 min  (longer incubations recover more RNA). 
Centrifuge the mixture at  . Dispose of the supernatant.
Wash the pellet by gently adding ice-cold 70% ethanol and centrifuge at  . Dispose of the supernatant.
Dry the pellet until the remaining ethanol has just evaporated. Drying for too long will impair subsequent resuspension. 
Resuspend in nuclease free water.