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Xenopus Whole Mount In Situ Hybridization

Nathalie Escande-Beillard, March 2009

ISH is a method used to determine the spatio-temporal expression pattern of a gene.

Synthesis of antisense RNA Probe:

1. Linearize DNA plasmid by digesting with a suitable enzyme. Check for complete
digestion by running 1µl of DNA on agarose gel.

**All conditions should be RNase free: use gloves and filtered tips.

2. Phenol:Chloroform extract and ethanol precipitate DNA or use a Kit for cleaning DNA.

3. Resuspend DNA in a suitable volume of RNase free water to get approximately 0.5 or
1µg/µl. Measure DNA concentration (nanodrop).

4. Set up transcription reaction:

1000-2000ng DNA
4µl 10X Transcription Buffer
4µl Labelling Mix (Digoxigenin)
1µl RNA guard (40 U/µl)
4µl RNA polymerase (20U/ µl)
RNAse free Water to 40µl

5. Incubate for 4-5 hours at 37⁰C.

6. Treat with DNase for 20mn at 37⁰C (1 unit of enzyme/µg DNA).

7. Remove unincorporated, free nucleotides with Quick Spin Columns:

• First remove top cap from column and then bottom cap to avoid air bubbles.
• Spin for 5 minutes at 4ºC, 1800 rpm in a swing bucket centrifuge.
• Remove eluate and spin for an additional 5 minutes.
• Put columns in new (labeled) tubes, add transcription reaction making sure to add
it directly on the center of the column while not disrupting resin with the pipette
tip.
• Spin for 15 minutes at 4ºC, 1800 rpm.

8. Quantify RNA yield with nanodrop to determine how much to use for the ISH.

9. Run a gel with 1µl to verify expected size and quality of probes
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General Notes:
¾ All recipes and most chemicals used are listed with their vendor and catalog number at the
end of this protocol.
¾ Very important to work in RNAse free conditions. Use gloves and filtered tips from the
fixation step to the end of hybridization.

Day 1:
¾ All steps in Day 1 are done ON ICE except for Proteinase K treatment (step #3).
¾ Use at least 2ml of buffer for each wash, unless indicated.

1. Rehydrate the embryos through a methanol series in PBSw (75%, 50%, 25%). Each
rehydration step is incubated for 5 minutes.

2. Wash 3X with PBSw, 5 minutes each.

3. Treat embryos for 8 minutes with 10µg/ml Proteinase K in PBSw at room temperature
(1ml per tube). Staining for highly expressed genes requires less digestion, but longer
digestion may help for genes with lower expression. Do not exceed 8 minutes !

4. Stop digestion by washing with 2mg/ml glycine in PBSw.

5. Do a fast wash with PBSw, and then wash 2X with PBSw, 5 minutes each.
 
6. Refix embryos in 5ml of 4% paraformaldehyde / 0.2% glutaraldehyde in PBSw for 15
minutes. Make the buffer fresh each time.

7. Do a fast wash with PBSw, and then wash 3X with PBSw, 5 minutes each.

8. Wash in 1ml of 50% PBSw / 50% Hybridization Solution for 3 minutes.

9. Wash in 1ml of Hybridization Solution (100%) for 3 minutes.

¾ Embryos can also be stored at this step in Hybridization Solution at –20ºC.

10. Replace 1ml Hybridization Solution with 400µl Hybridization Solution and prehybridize
for 3 hours at 65ºC.

11. Denature appropriate amount of probe in 100µl Hybridization Solution at 95ºC for 5
minutes. Put on ice, vortex and add this mix to the embryos and hybridize overnight at
70ºC.
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Day 2:
1. Remove probe/hybridization mix and replace with 800µl Hybridization Solution. Wash
for 5 minutes at 70ºC.

2. Add 400µl 2x SSC (pH4.5) to each vial. Incubate 5 minutes at 70ºC.

3. Repeat the previous step 2 more times (final volume = 800µl + 400µl + 400µl + 400µl =
2000µl). Incubate at 70ºC for 5 minutes each time.

4. Remove the mix and wash 2X with 2x SSC (pH7) / 0.1% CHAPS at 70ºC for 30 minutes.

5. Wash 2X in MAB at room temperature for 10 minutes each.

6. Wash 2X in MAB at 70ºC for 30 minutes each.

7. Wash 2X in PBS at room temperature for 10 minutes each.

8. Wash in PBSw at room temperature for 5 minutes.

9. Incubate the embryos in 1ml Antibody Buffer (without antibody) at 4ºC, rocking, for a
minimum of 2 hours.

10. At this time also pre-block the antibody in Antibody Buffer at 4ºC, rocking for 2 hours:
Anti-Dig - Alkaline Phosphatase dilute 1:5000 from a stock of 150 units/200ul.
Anti-Dig – Peroxidase dilute 1:200 from a stock of 150 units/ml.

11. Replace Antibody Buffer with 1.5ml of pre-blocked antibody. Incubate with rocking at
4ºC overnight. Check embryos to make sure all are immersed in solution (not stuck in lid
of glass).

Day 3:

1. Fast wash embryos with 0.1% BSA in PBSw.

2. Wash 5X in 0.1% BSA in PBSw, with rocking, 1 hour each wash at room temperature.

3. Wash 2X in PBSw for 30 minutes each, at room temperature.

4. Wash 2X in AP1 Buffer for BM Purple staining for 10 minutes each.

5. Replace AP1 Buffer with 1ml BM Purple, cover with Aluminium foil and incubate with
rocking until desired staining is reached. Check embryos to make sure all are immersed
in solution (not stuck in lid of glass). Staining time will vary depending on the level of
expression and probe quality. It is recommended to let the reaction take place at 4⁰C,
overnight. At room temperature embryos will tend to get more background.
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Day 4:

1. Stop staining reaction by washing 2 times in Stop Solution for 15 minutes each. Rinse
caps as well.

2. Dehydrate through a methanol series (25%, 50%, 75%, 2x 100%). Embryos can be stored
in methanol at -20ºC.

3. Optional: to remove pigmentation or excess background and to enhance contrast,

embryos can be bleached with 3ml of fresh bleaching solution. To speed up the process,
place tubes on aluminium foil and under intense neon light several hours to overnight.

4. Put back embryos in methanol 100 %.

Solutions:

¾ When diluting 10x stock solution use DEPC treated water and filter.
¾ All other buffers are made using DEPC treated water and then filter.

DEPC treated buffers or water

Add 0.1% DEPC
Incubate with agitation until it is completely dissolved and autoclave.

10x PBS
80g NaCl
2g KCl
14.4g Na2HPO4
2.4g KH2PO4
800ml distilled Water (DDW)
Dissolve, pH to 7.4, add DDW to 1L, DEPC treat and autoclave.
 
PBSw
PBS with 0.1% Tween-20
DEPC treat and autoclave.

20x SSC
175.3g NaCl
88.2g Sodium Citrate
800ml DDW
Dissolve, pH to 7.0, add DDW to 1L, DEPC treat and autoclave.

4% Paraformaldehyde
Dissolve paraformaldehyde in fresh PBS (4g for 100ml).
Heat at 60ºC and mix until completely dissolved.
Cool on ice, filter, aliquot and store at - 20⁰C
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Hybridization Solution
¾ Make 1L, filter, aliquot and store at –20ºC.
10g Boehringer Block
500ml Formamide
250ml 20x SSC
¾ Heat at 65ºC for 1 hour
120ml DEPC treated water
100ml Torula RNA (10mg/ml in water; filtered)
2ml Heparin (50mg/ml in 1x SSC)
5ml 20% Tween-20
10ml 10% CHAPS
10ml 0.5M EDTA

MAB
100mM Maleic Acid
150mM NaCl pH 7.5

Boehringer blocking solution (10%)

Dissolve Boehringer blocking reagent in maleic acid buffer (MAB) (10g for 100ml).
Heat and vortex frequently to dissolve completely.
Store at –20°C as a stock solution.

Antibody Buffer
10% heat inactivated goat serum
10% Boehringer blocking stock solution.
80% PBSw
Heat at 70°C for 10 minutes, vortex, cool on ice and filter.
Aliquot and store at –20°C.

AP1 Buffer
0.1M NaCl
0.1M Tris pH 9.5
50mM MgCl2

Stop Solution
100mM Tris pH 7.4
1mM EDTA

Bleaching solution
2/3 Methanol 100%
1/3 Hydroxyde Peroxyde 31.5% (final 10.5%)
Make fresh each time.
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Chemicals for making the probe:

Dig RNA Labelling Mix (10x) – Roche #1277073
Flu RNA Labelling Mix (10x) – Roche #1685619
Transcription Buffer (10x) – Roche #1465384
RNA Guard – Roche #3335399
T3 RNA Polymerase – Roche #1031163
T7 RNA Polymerase – Roche #881767
SP6 RNA Polymerase – Roche #810274
Quick Spin Columns – Roche #1274015
QIAquick Gel Extraction Kit – Qiagen #28706

Chemicals for ISH:

Boehringer Block – Roche #1096176
Proteinase K – Gibco #25530-049
Anti Dig-AP – Roche #1093274
Anti Flu-AP – Roche #1426338
Anti Dig-POD – Roche #1207733
Anti Flu-POD – Roche #1426346
BM Purple – Roche #1442074
Hydrogen Peroxide 31.5% – Calbiochem#386790
 

 
 
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