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INFORMATION FOR

In Situ Hybridization - Detailed Protocol

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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 substituted ribonucletide for this in situ procedure.

Protocol for Making Antisense RNA probe (dig labelled)

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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%Tween-20)
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 RTAcetic 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) RTRefix 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
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.
___ 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.


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/mlThe 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 (1/3000dilution of the anti-digoxygenin AP antibody) overnight at 4°C (or 4 hrs at RT.) 0.2 µL/0.5 mL

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 ml 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 ml/ml NBT in AP buffer

3.5 ml/ml BCIP in AP buffer

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.

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 MABRTThe color reaction is stopped by a change in pH
___ Fix overnight in Bouin's RTThe Bouin's also stops the chromogenic reaction and also stabilizes the stain in the embryos.
___ Multiple 10 minute washes in 70% buffered EtOH RTRemoves 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 in BB:BA clearing agent (benzyl benzoate/benzyl alcohol).BB:BA nicely clears the embryo but requires that the
embryo be dehydrated first. To dehydrate, place embryos
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 concentrationfor 100 mL

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

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). Corrections thanks to Vivian Choi and Neha Prakash.

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.