Cochlea
Whole mount in situ hybridization on mouse cochleas
A
B
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.
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Cochlea
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
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Cochlea
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
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Cochlea
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 m, depending on the staining.
Mounting slides after cryosectioning
2 possibilities: mounting with water-based mounting media or with xylenebased 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
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Cochlea
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
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Cochlea
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
20x SSC DEPC, pH7
Blocking Solid
Heparin 50 mg/ml
5 ml
2.5 ml
200 mg
10 µl
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Cochlea
100x Triton
10% Chaps
EDTA 0.5M DEPC
tRNA
H2O DEPC
10 µl
100 µl
100 µl
10 mg
2070 µl
10 ml
KTBT
Tris 1M pH 7.5
NaCl 5M
KCl 3M
100x Triton
H2O DEPC
25 ml
15 ml
1.7 ml
5 ml
453 ml
500 ml
NTMT
Tris 1M pH 9.5
NaCl 5M
MgCl2 1M
100x Triton
H2O DEPC
50 ml
10 ml
25 ml
5 ml
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
g of clone is needed and linearized on each side of the insert to generate the
sense and antisense probes.
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Cochlea
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
ddH20
NTP labeling mix
Cleaved plasmid
RNAse inhibitor
1.
2 µl
X µl
2 µl
1 µg
1 µl
20 µl
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.
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Cochlea
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.
For questions, contact Dr. Sarah Vreugde.
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