xenopuswholemountinsituhybridization

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Xenopus Whole MountIn 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 completedigestion by running 1l 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

1g/ l. Measure DNA concentration (nanodrop).

4. Set up transcription reaction:1000-2000ng DNA

4l 10X Transcription Buffer

4l Labelling Mix (Digoxigenin)

1l RNA guard (40 U/l)4l RNA polymerase (20U/ l)

RNAse free Water to 40l

5. Incubate for 4-5 hours at 37C.6. Treat with DNase for 20mn at 37C (1 unit of enzyme/g 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 4C, 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 4C, 1800 rpm.8. Quantify RNA yield with nanodrop to determine how much to use for the ISH.9. Run a gel with 1l to verify expected size and quality of probes


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General Notes:

All recipes and most chemicals used are listed with their vendor and catalog number at theend of this protocol.

Very important to work in RNAse free conditions. Use gloves and filtered tips from thefixation step to the end of hybridization.

Day 1:

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

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

2. Wash 3X with PBSw, 5 minutes each.3. Treat embryos for 8 minutes with 10g/ml Proteinase K in PBSw at room temperature

(1ml per tube). Staining for highly expressed genes requires less digestion, but longerdigestion 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.

Embryos can also be stored at this step in Hybridization Solution at 20C.10.Replace 1ml Hybridization Solution with 400l Hybridization Solution and prehybridize

for 3 hours at 65C.

11.Denature appropriate amount of probe in 100l Hybridization Solution at 95C for 5minutes. Put on ice, vortex and add this mix to the embryos and hybridize overnight at

70C.


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Day 2:

1. Remove probe/hybridization mix and replace with 800l Hybridization Solution. Washfor 5 minutes at 70C.

2. Add 400l 2x SSC (pH4.5) to each vial. Incubate 5 minutes at 70C.3. Repeat the previous step 2 more times (final volume = 800l + 400l + 400l + 400l =

2000l). Incubate at 70C for 5 minutes each time.

4. Remove the mix and wash 2X with 2x SSC (pH7) / 0.1% CHAPS at 70C for 30 minutes.5. Wash 2X in MAB at room temperature for 10 minutes each.6. Wash2X in MAB at 70C 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 4C, rocking, for a

minimum of 2 hours.

10.At this time also pre-block the antibody in Antibody Buffer at 4C, 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 at4C 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 4C,

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. Rinsecaps as well.

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

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:

When diluting 10x stock solution use DEPC treated water and filter. 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 60C and mix until completely dissolved.

Cool on ice, filter, aliquot and store at - 20C


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Hybridization Solution

Make 1L, filter, aliquot and store at 20C.10g Boehringer Block

500ml Formamide

250ml 20x SSC

Heat at 65C for 1 hour120ml DEPC treated water100ml 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 20C as a stock solution.

Antibody Buffer

10% heat inactivated goat serum

10% Boehringer blocking stock solution.80% PBSw

Heat at 70C for 10 minutes, vortex, cool on ice and filter.

Aliquot and store at 20C.

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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xenopuswholemountinsituhybridization

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