Whole Mount In Situ Hybridization
Protocol
Purpose of in situs: To use an RNA probe to stain for the specific complementary mRNA of an intact
embryo, allowing visualization of mRNA patterns.
Note: When making in situ probe, use digoxigenin substituded ribonucletide for this in situ procedure.
Fixation of embryos before in situ for long term storage:
Fix embryos in MEMFA for 1-2 hr (room temp): 1xMEMFA: 1/10 vol 10xMEMFA salt
1/10 vol formaldehyde
8/10 vol water
Dehydrate in MeOH (make sure they are completely dehydrated by changing MeOH several times)
Embryos can now be stored long term at –20°C or go straight to in situs
Note: Use of albino embryos allows one to skip bleaching step
Pre-in situ preparations: Collect the embryos at the correct stage and aliquot them into baskets with mesh
screening. Place mesh screen baskets into basket holder and place basket holder into a wash container in
which all washes will take place. It is useful to have two wash containers so one can prepare the
subsequent wash ahead of time and simply transfer entire basket holder into second wash container.
Begin In Situ
DAY 1: ~3 hours needed until 6 hr. prehyb step
Reagents:
Thaw paraformaldehyde 20% (10-20 ml)
Set water bath to 60oC (shaker)
Make 1 Liter 1xPBS+0.1% Tween-20 (PTw) (100 ml 10xPBS+1ml Tween-20. Add H20 to 1 L)
Thaw Proteinase K 10mg/ml (50-100 µl)
Methanol
0.1M Triethanolamine pH 7-8 (~100-200 ml)
Acetic Anhydride (125-250 µl)
Hyb Buffer (50-100 ml)
Rehydration: (PTw=1xPBS+0.1%Tween20)
5 minutes each wash each RT (room temp)
Methanol (MeOH)
75% MeOH 25% H2O
50% MeOH 50% H2O
25% MeOH 75% PTw
100% PTw
3x 5 minutes in PTw RT
1) 
2) 
3) 
Rehydration into PTw from MeOH is done gradually to
prevent the formation of bubbles in embryos. PTw is used
because the detergent (Tween) prevents the embryos
from sticking to each other. Furthermore, PTw is a
phosphate buffered saline solution that helps to neutralize
the negative charge of mRNA so that the probe can bind
better to it. When preparing MeOH and water washes: if
using small glass container, use a total of 40 mL, or if
using big glass container, use a total of 80 mL. Use
graduated cylinder to measure out and mix reagents. For
example, 75% MeOH 25% water – use small glass
container and combine 30 mL MeOH and 10 mL water
 Proteinase K ~5 minutes RT
(100µl of 10mg/ml solution PK/100ml PTw)
The purpose of this step is to permeabilize the embryo by
removing some of the outer proteins which allows better
probe penetration for deeper tissues. This step can be
omitted if the staining is restricted to superficial tissues.
Proteinase treatment requires careful monitoring and
should not be prolonged.
Rinse 2x 5 minutes in 0.1M triethanolamine
pH 7-8 RT
1) 
2) 
The triethanolamine is a buffer without free amines which
is necessary for the next step.
Add 12.5µl acetic anhydride/5mls: 5
minutes (125µl in 50 ml/250µl in 100 ml) - in
triethanolamine RT
Acetic anhydride acetylates free amines to neutralize
positive charge so that the probe will bind specifically to
mRNA rather than nonspecifically due to electrostatic
interactions. The acetic anhydride is sensitive to water
vapor in the air, so be sure to pipette it only right before
you will be using it. Keep lid on acetic anhydride closed
at all times..
Repeat acetic anhydride addition: 5
minutes RT
Wash 2x 5 minutes in PTw
1) 
2) 
Refix for 20 minutes with 4%
paraformaldehyde in PTw (10 ml 20% PF
into 40 ml PTw RT
Refix the embryo to insure embryo integrity after the PK
step.
Wash 5x 5 minutes in PTw (wash off
excess paraformaldehyde) RT
1) 
2) 
3) 
4) 
5) 
Prehybridize 1 hour for trops or 6 hours
for laevis 60°C. (save prehyb solution on
your bench)
See appendix for hybridization buffer preparation
Replace with 0.5ml probe solution
(1µg/ml probe), hybridize overnight at 60°C.
Place probe into specific tubes that fit basket exactly.
Baskets in Falcon 2059 tubes work nicely.
Save the pre-hyb buffer for the following day at RT.
Prehyb buffer has many different components to help
keep RNA denatured so that is it linear and can bind to
probe effectively. It also has other components that
minimize non-specific binding.
Day 2: ~4 hours
Reagents:
2xSSC (100 ml 20xSSC into 900 ml H2O)
Thaw RNAse A (20 mg/ml)/RNAse T1 (10 mg/ml) (Both at 1000x)
Thaw MAB + 10% BMB Blocking agent (5-10 ml)
2xSSC with RNase A 20 µg/ml, RNAse T1 10 µg/ml
0.2xSSC (100 ml 2xSSC into 900 ml H2O)
MAB
Remove probe and keep for reuse
Replace probe with hybridization buffer
(use prehyb. solution from previous step),
Incubate 60°C for 3 minutes
3 minutes 2xSSC, 60°C
3 minutes 2xSSC, 60°C
Transfer baskets to basket holder.
Make sure you don't go over 10 minutes incubation
because otherwise the probe might migrate to the next
basket and to adjacent embryos.
Washes
3x 20 minutes 2xSSC, 60°C
1) 
2) 
3) 
Remove excess hybridization buffer and excess probe.
Wash 30 minutes (37oC) 2x SSC with
RNase A 20 µg/ml, RNAse T1 10 µg/ml
The RNAse A and T1 degrade mismatched double
stranded RNA.
Wash once in 2x SSC 10 minutes RT
Wash twice in 0.2x SSC 30 minutes, 60°C.
1) 
2) 
Wash away RNAse
Wash twice in MAB, 10-15 minutes, RT
1) 
2) 
 Wash in MAB + 2% BMB Blocking
Reagent (10 ml 10% BMB/1xMAB to 50 ml
with MAB) 1 hr or more, RT.
BMB is a blocking reagent to prevent non-specific
antibody binding
Replace with MAB + 2% BMB Blocking
Reagent + Ab (1/3000 dilution of the antidigoxygenin AP antibody) overnight at 4°C
(or 4 hrs at RT.) 0.2 µL/0.5 mL
Antibody with alkaline phosphotase – BM purple removes
a phosphate and turns the substrate purple
mRNA
DAY 2.5-3 (5.5 hours)
Reagents:
Thaw Alkaline Phosphatase buffer (with 0.005 M levamisol and 0.1% Tween) (100-200 ml)
MAB pH~7.0
Wash embryos at least 5 times, 1 hour each
with MAB RT
1) 
2) 
3) 
4) 
5) 
These washes are necessary in order to wash off excess
antibody. These washes are extremely flexible. Often
times we do one quick wash and then let it wash
overnight at 4oC. Occasionally we have left the embryos
in MAB over the weekend at 4oC and have gotten good
results. However, longer than 3-4 days in MAB does
tend to lead to weaker staining.
Wash twice, 5 minutes each at room
temperature with Alkaline phosphatase
buffer (make sure levamisol 0.005 M and
0.1% Tween have been added)
1) 
2) 
This buffer inhibits endogenous phosphatases.
Replace last wash with BM purple.
500 l of BM purple per basket. Wrap in
aluminum foil.
NBT/BCIP is an effective alternative and
may have higher resolution
NBT – 75 mg/ml in 70% DMF
BCIP – 50 mg/ml in 100% DMF
4.5 l/ml NBT
3.5 l/ml BCIP
BM purple turns blue and precipitates in the presence of
the antibody conjugated to alkaline phosphatase. The
crispest and most accurate stain occurs when the stain
develops very quickly and then is stopped. In this
situation diffusion of stain is minimized. However you will
need to tailor the staining step for your particular needs.
We typically monitor the stain at RT every half hour and
many probes will be visualized nicely in 2-4 hours.
Alternatively the embryos can be left at 4oC overnight
with the stain developing more slowly. Rarely the
embryos will be overstained at 4oC o/n but not typically.
BM
Purple
BM
Purple
Phosphate
Group
Yellow
Purple
The alkaline phosphatase will remove the
phosphate group from BM purple.
Stopping Color Reaction:
Reagents:
MAB (malaeic acid buffer)
Bouin's Fixative (70 ml saturated Picric acid/5 ml glacial acetic acid/25 ml formaldehyde)
1xSSC (1 liter)
EtOH
Bleach Solution (1% H2O2, 5% formamide, 0.5x SSC)
20xSSC
formamide
30% peroxide
 Stop chromogenic reaction with quick
wash in MAB RT
The color reaction is stopped by a change in pH.
 Fix overnight in Bouin’s RT
The Bouin’s also stops the chromogenic reaction and
also stabilizes the stain in the embryos.
Multiple 10 minute washes in 70%
buffered EtOH RT
Removes yellow Bouin’s. Do washes until the embryos
become white again.
Rehydrate stepwise into 1X SSC: RT
5 min 75% buffered EtOH: 25% 1X SSC
 5 min 50%:50%
 5 min 25%: 75%
 5 min 100% 1xSSC
 5 min once more in 1xSSC
Bleach embryos in bleaching solution (~1-2 hours RT): for 50 mls:
1.25 ml 20xSSC
2.5
ml formamide
2
ml 30% peroxide
45
ml distilled water
In order for the bleaching reaction to work, put embryos under direct light. Use light box.
Wash twice for 5 min in 1xSSC RT
6) 
7) 
Take pictures of uncleared or clear embryos BB:BA nicely clears the embryo but requires that the
in BB:BA clearing agent (benzyl
embryo be dehydrated first. To dehydrate, place embryos
benzoate/benzyl alcohol).
immediately in MeOH. There is no need to do a gradual
dehydration. Do multiple changes of MeOH to insure that
they are fully dehydrated.
Embryos can be stored in 80% glycerol at RT which also
partially clears embryos. Just add 100% glycerol to
dehydrated embryos. The MeOH will evaporate and the
embryos will clear a little. Embryos can also be stored in
70% ethanol or methanol at low temperature (-20oC).
Appendix
10x MEMFA salts
1 M MOPS
20mM EGTA
10mM MgSO4
10x salts can be autoclaved and stored. Turns yellow after autoclaving.
Hybridization buffer:
Final concentration
50% formamide
5x SSC
1 mg/ml Torula RNA
100 µg/mL heparin
1x Denhart’s
0.1% Tween 20 (Sigma P-1379)
0.1% CHAPS (Sigma C3023)
10 mM EDTA
for 100 mL
50 mL of formamide
25 mL of 20xSSC
2 mL of 50 mg/mLTorula RNA
10 mg heparin
1 mL 100xDenhart’s
0.1 ml Tween 20
0.1 gm CHAPS
5 mL 0.2 M EDTA
MAB
100 mM maleic acid (Sigma M0375)
150 mM NaCl
Alkaline Phosphatase Buffer
100 mM Tris (pH 9.5)
50 mM MgCl2
100 mM NaCl
0.1% Tween 20 (Sigma P1379)
2 mM levamisol (Sigma)
We keep aliquots frozen at –20 with levamisol.
Contributed by Joanna Yeh and Mustafa K. Khokha. Protocol based on (Sive et al 2000)
References
Sive, HL, Grainger RM, Harland RM; Early Development of Xenopus Laevis: A Laboratory Manual; Cold
Spring Harbor Laboratory Press; New York, 2000; pp. 249-297.