Whole Mount in Situ Hybridization on Mouse Cochleas

Cochlea

Whole mount in situ hybridization on mouse cochleas

Ush3a expression in the hair cells of a p0 mouse. (A) Whole

mount cochlea (left, antisense probe; right, sense probe.

(B) Antisense probe, cut as shown by line in A.

Principle

In situ hybridization is used to localize mRNA to a specific cell type. In order to do this, a region specific for the gene of interest is in vitro transcribed using the Digoxigenin (DIG) labeling kit (Roche Molecular Biochemicals), which is a non-radioactive way of labeling the probe. This method is known to be very specific and very sensitive, even more than the radioactive method of labeling. Once the probe is prepared, a whole mount in situ hybridization experiment can be carried out on cochlear ducts. The easiest and best results are usually with cochleas between p0 and p5. For embryos, this method works best from e7.5-11.5. After the experiment, individual expression patterns in certain cell types must be revealed by sectioning the cochleas, which we perform by cryosectioning.

Whole mount in situ hybridization on mouse cochleas (or embryos)

From A. Nieto with modifications.

1. Dissect cochleas in PBS.

2. Incubate in 4% paraformaldehyde in PBS at 4°C for 2 hrs to on.

3. Rinse with PBT twice for 5 min.

4. Dehydrate by washing for 10 min each in a graded methanol series diluted in PBT (25% methanol, 50% methanol, 75% methanol) and then twice in 100% methanol. The cochleas can be stored at 20°C for several months.

5. Rehydrate cochleas by washing in a graded methanol series in PBT (75% methanol, 50% methanol, 25% methanol) and then twice in PBT.

6. Treat the cochleas with 20 µg/ml proteinase K in PBT for 5-10 min at rt.

7. Rinse with PBT twice for 5 min.

8. Refix the cochleas with fresh 0.2% glutaraldehyde/4%paraformaldehyde in PBT for 20 min.

9. Rinse twice with PBT for 5 min.

10. Remove most of the liquid, add prewarmed prehybridization solution (60°C) for 2 hr to on.

11. Remove prehybridization solution and add hybridization mix. Incubate on up to 3 nights/2 days, 60°C, shaking.

12. Wash with 2xSSC, 0.1% CHAPS, 3 times for 20 min at 60°C.

13. Wash with 0.2xSSC, 0.1% CHAPS, 3 times for 20 min at 60°C.

14. Rinse with KTBT twice for 10 min, rt.

15. Preblock the cochleas with 2%NGS, 1%Block solution in KTBT for 3 hrs at rt or 4°C on.

16. Incubate with 1/500- 1/2000 dilution of anti-DIG in 1%NGS/1% block solution in KTBT, on, 4°C.

17. Wash in KTBT rt for 1hr >5 times, then on at 4°C.

18. Wash twice in NTMT for 15 min and transfer to a dish for observation.

19. Incubate in the dark with NTMT containing NBT/BCIP (10 µl NBT/BCIP /500 µl NTMT). Periodically monitor the reaction, and when a strong reaction is produced and/or background is seen, stop the reaction by washing several times with KTBT. Store on or more in KTBT, 4°C.

20. Reevaluate the reaction; when stronger staining is wanted, wash 2 times in NTMT and incubate again with 10 µl NBT/BCIP /500 µl NTMT. Stop reaction by washing with KTBT. The cochleas can be kept up to 1 month in KTBT, 4°C.

Details of the protocol, step by step

General: all steps (from2-18) are done with gentle rocking on a rocking platform. The experiment is carried out in 2 ml eppendorf tubes, and solutions are changed by removing most of the liquid, and putting in the fresh solutions. Try not to remove the whole solution at any time (there should always be some solution covering the cochleas).

Dissection: the initial dissection, step1, should be fast, and dissection should be just so the PFA can easily reach all areas of the cochlear ducts. For older cochleas (p7 and older), pierce the oval and round windows, and make a hole in the apex. No decalcification is needed for cochleas up to p10. For younger cochleas (e16-p7), take out cochlea from its cartilaginous/bony cochlea, or make big holes in the covering shell. For e12 and e14 cochleas, fixation is good when cochlea is removed as a whole with surrounding tissues. The next dissection step should be after rehydration, in PBT, step 5. In this dissection step, remove parts of the stria vascularis in the 2-3 turns of the cochlea, but make sure not to remove the whole stria, as the cochleas then tend to collapse. The final dissection should only be after the whole procedure.

Step 6: proteinase K treatment. Prepare the proteinase K fresh from a stock. The time of the treatment is critical.

Step 19. NBT/BCIP needs to be mixed very well. Incubate for 10 min at 37°C, vortex.

Decalcification

Many surgical specimens contain calcified areas that need to be decalcified before processing and sectioning. This is achieved as follows.

1. When the specimen is sufficiently fixed, the selected tissue is placed into a labelled pot of 8% formic acid and left on the shelf until the following day.

2. Each day thereafter the tissue is assessed until it is deemed soft enough to section after processing.

3. If it is not ready, the decalcifying fluid is changed and the pot is replaced onto the shelf for a further 24 hrs.

4. This procedure is repeated daily until decalcification is complete.

In principle, decalcification as such can be done for whole mount cochleas after p10, and takes 1-3 days. We usually perform the decalcification after step 4 and use 8% formic acid in methanol, which can be kept at 20°C for this period of time. It makes the subsequent dissection much easier, but the in situ reaction is less sensitive.

Another way to decalcify is with EDTA. We have no experience with this, but it is used and may be better.

Cryosectioning of cochleas after whole mount in situ hybridization

After taking pictures of the whole mount cochleas:

1. 10% sucrose in PBS, 1 hr, rocking, rt.

2. 20% sucrose in PBS, 1 hr, rocking, rt.

3. 30% sucrose in PBS, on, rocking, 4°C.

4. 1/2 30% sucrose in PBS/ 1/2 OCT compound, on.

5. OCT compound 1-2 hrs.

6. Fresh OCT compound, orient cochleas and freeze in microbeakers on dry ice.

Comments:

· When time is limited, step 4 can be after >1hr incubation in 30% sucrose. As a general rule, cryoprotection in sucrose is complete when the tissue sinks in a 30% sucrose solution.

· To orient the cochlea, keep in mind that the cuts will go parallel to the bottom of the beaker. When you want to cut a certain area of a cochlea, it is best to dissect out the area of the cochlea for proper orientation. If it is not critical which area is cut, simply place the cochlea on its side with the axis going through the modiolus parallel to the bottom of the beaker.

· Thickness of the cuts should be 6 - 14 mm, depending on the staining.

Mounting slides after cryosectioning

2 possibilities: mounting with water-based mounting media or with xylene-based mounting media. Xylene-based mounting is considered better, without fading of the results after some time. The major disadvantage is that sections of small entities, such as a whole mount cochlea, tend to "slide off" the slide during the dehydration in ethanol, even with the best possible slides. Therefore, for small sections, use water based mounting media.

Slides: Super-Frost plus

Mounting media: water-based: GelMount

xylene-based: Permount or equivalent

Hematoxylin counterstaining

1. Fix in 70% ethanol.

2. Wash (by dipping) in ddH20 3 times.

3. Hematoxylin >45 s.

4. Wash in ddH20 3 times.

5. Wash in tap water, 5 min.

6. 50% ethanol, 2 min.

7. 75% ethanol, 2 min.

8. 100% ethanol, 2 min.

9. 1/2 100% ethanol/1/2 xylene, 1 min.

10. Mount with Permount.

For mounting with xylene-based mounting media, without counterstaining, perform only steps 6-10.

Practically: put some drops of mounting medium on the sections and place a cover slip on them. Leave overnight horizontally to dry.

Solutions

4% PFA

4 g PFA in 100 ml + 5 drops 5 N NaOH; dissolve at 55°C in water bath, stir occasionally; adjust pH to 7.0 -7.5 (2 drops 37% HCl [12.5 N], and check pH

Some use pH 9.5 for in situ hybridization. Both pH values work for us.

PFA 8% stock in ddH20 can be frozen (-20°C), in aliquots.

For use, make 4% PFA in PBS or PBT by taking 1/2 8% PFA in ddH20 and 1/2 2x PBS(T).

30% sucrose in PBS

30 g in 100 ml PBS; store at 4°C

DEPC-treated PBS, ddH20.

Add DEPC to a final concentration of 0.1% (500 µl in 500 ml) v/v, incubate at 37°C for 2 hrs , dark, autoclave.

PBS DEPC

From stock 40 ml 10x PBS, 360 ml ddH20, 400 µl DEPC, incubate at 37°C for 2 hrs, dark, autoclave.

PBT DEPC

PBS DEPC, 0.1% Triton x-100

2x PBS DEPC

From stock 80 ml 10x PBS, 320 ml ddH20, 400 µl DEPC, incubate at 37°C for 2 hours, dark, autoclave.

Proteinase K

Stock 10 mg/ml

To make the stock, add 9 ml DEPC ddH20 to 90 mg bottle to make 10 mg/ml. Freeze -20°C, in aliquots.

To treat the cochleas, use 20 µl/ml

20 µl/ml proteinase K: 2 µl of stock, add 998 µl PBT

Heparin, NGS, CHAPS 10%, Proteinase K stock, blocking solution are frozen in aliquots in -20°C.

Probe

Use between 40-50 ng/ml to 400-500ng/ml prehybridization solution. We usually work with 100-200 ng/ml.

Prehybridization solution

Formamide x 100 / 5 ml
20x SSC DEPC, pH7 / 2.5 ml
Blocking Solid / 200 mg
Heparin 50 mg/ml / 10 µl
100x Triton / 10 µl
10% Chaps / 100 µl
EDTA 0.5M DEPC / 100 µl
tRNA / 10 mg
H2O DEPC / 2070 µl
10 ml

KTBT

Tris 1M pH 7.5 / 25 ml
NaCl 5M / 15 ml
KCl 3M / 1.7 ml
100x Triton / 5 ml
H2O DEPC / 453 ml
500 ml

NTMT

Tris 1M pH 9.5 / 50 ml
NaCl 5M / 10 ml
MgCl2 1M / 25 ml
100x Triton / 5 ml
H2O DEPC / 410 ml
500 ml

Probe preparation

As a rule of thumb, the probe should have a length of over 400 bp, and the results tend to be better with longer probes of up to 2000 bp. This region of the gene of interest should be specific for this gene, should not contain any repetitive pieces of sequence, and when blunt-end ligation to Bluescript is intended, should not contain an SrfI restriction enzyme site. To determine restriction enzyme sites this, the following web sites can be used:

http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, using the corresponding protein sequence;

http://ftp.genome.washington.edu/cgi-bin/RepeatMasker; use html as return format;

http://www.firstmarket.com/cutter/cut2.html.

This piece is then cloned into a vector suitable for in vitro transcription such as PCR-Script Amp Cloning vector (Bluescript), polished, ligated, and transformed into ultracompetent cells. For the in vitro transcription, about 30 mg of clone is needed and linearized on each side of the insert to generate the sense and antisense probes.

Linearize ~30 µg vector by restriction enzyme cleavage.

Run on agarose gel and extract linearized product.

Purification of plasmid

1. Add one volume of phenol/chloroform to one volume of cleaved plasmid, vortex for 20 s.

2. Centifuge at 13000 rpm for 10 min.

3. Transfer the upper phase to a new tube. For 20 µl DNA solution, add 10 µl (3M NaAc) and 70 µl (100% ethanol). (As a general rule, the final EtOH concentration has to be 70% and the final salt concentration has to be 0.3 M.

4. Place at -80°C for 20 min, or on at -20°C.

5. Centrifuge at 13000 rpm for 10 min, 4°C.

6. Wash pellet in cold 70% EtOH.

7. Centrifuge at 13000 rpm for 10 min, 4°C.

8. Air dry for ~10 min (until no fluid apparent, but not too long!)

9. Dissolve pellet in a proper volume of RNAse free ddH20. (Note: it is reported that DEPC can inhibit RNA polymerase; we have not experienced this problem).

In vitro transcription of probe

Using DIG RNA Labeling Kit (Roche Molecular Biochemicals)

10x transcription buffer / 2 µl
ddH20 / X µl
NTP labeling mix / 2 µl
Cleaved plasmid / 1 µg
RNAse inhibitor / 1 µl
20 µl

1. Centrifuge briefly and let stand for 2 hrs at 37°C.

2. Stop reaction by adding 2 µl 0.2 M EDTA pH8.

3. Add 2.5 µl 4M LiCl and 75 µl 100% ice cold EtOH.

4. Let stand for 30 min at -80°C.

5. Spin down for 10 min at 13000rpm, 4°C.

6. Wash pellet in 70% EtOH.

7. Spin down for 10 min at 13000rpm, 4°C.

Let dry about 10 min-30 min at rt.

8. Dissolve pellet in ddH20 + 1 µl RNAse inhibitor

9. Let stand for 30 min at 37°C.

Run an aliquot on an agarose gel at 80 V to check integrity of probe. Aliquot and store at -80°C.