Whole Mount In Situ Hybridization Protocol
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1 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 Thaw paraformaldehyde 20% (10-20 ml) Set water bath to 60 o C (shaker) Make 1 Liter 1xPBS+0.1% Tween-20 (PTw) (100 ml 10xPBS+1ml Tween-20. Add H 2 0 to 1 L) Thaw Proteinase K 10mg/ml ( µl) Methanol 0.1M Triethanolamine ph 7-8 (~ ml) Acetic Anhydride ( µl) Hyb Buffer ( ml) Rehydration: (PTw=1xPBS+0.1%Tween- 20) 5 minutes each wash each RT (room temp) Methanol (MeOH) 75% MeOH 25% H 2 O 50% MeOH 50% H 2 O 25% MeOH 75% PTw 100% PTw 3x 5 minutes in PTw RT 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
2 should not be prolonged. Rinse 2x 5 minutes in 0.1M triethanolamine ph 7-8 RT 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 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 4) 5) Prehybridize 1 hour for trops or 6 hours for laevis 60 C. (save prehyb solution on your bench) Replace with 0.5ml probe solution (1µg/ml probe), hybridize overnight at 60 C. 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. 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.
3 Day 2: ~4 hours 2xSSC (100 ml 20xSSC into 900 ml H 2 O) Thaw RNAse A (20 mg/ml)/rnase T 1 (10 mg/ml) (Both at 1000x) Thaw MAB + 10% BMB Blocking agent (5-10 ml) 2xSSC with RNase A 20 µg/ml, RNAse T 1 10 µg/ml 0.2xSSC (100 ml 2xSSC into 900 ml H 2 O) 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 Washes 3x 20 minutes 2xSSC, 60 C Wash 30 minutes (37 o C) 2x SSC with RNase A 20 µg/ml, RNAse T 1 10 µg/ml Wash once in 2x SSC 10 minutes RT Wash twice in 0.2x SSC 30 minutes, 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. Remove excess hybridization buffer and excess probe. The RNAse A and T 1 degrade mismatched double stranded RNA. Wash away RNAse Wash twice in MAB, minutes, RT 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
4 DAY (5.5 hours) Thaw Alkaline Phosphatase buffer (with M levamisol and 0.1% Tween) ( ml) MAB ph~7.0 Wash embryos at least 5 times, 1 hour each with MAB RT 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 4 o C. Occasionally we have left the embryos in MAB over the weekend at 4 o C 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 M and 0.1% Tween have been added) This buffer inhibits endogenous phosphatases. Replace last wash with BM purple. 500 µl 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 µl/ml NBT 3.5 µl/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 4 o C overnight with the stain developing more slowly. Rarely the embryos will be overstained at 4 o C o/n but not typically. BM Purple Yellow Phosphate Group Purple BM Purple The alkaline phosphatase will remove the phosphate group from BM purple.
5 Stopping Color Reaction: 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% H 2 O 2, 5% formamide, 0.5x SSC) 20xSSC formamide 30% peroxide Stop chromogenic reaction with quick wash in MAB RT Fix overnight in Bouin s RT Multiple 10 minute washes in 70% buffered EtOH RT The color reaction is stopped by a change in ph. The Bouin s also stops the chromogenic reaction and also stabilizes the stain in the embryos. 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 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 (-20 o C).
6 Appendix 10x MEMFA salts 1 M MOPS 20mM EGTA 10mM MgSO 4 10x salts can be autoclaved and stored. Turns yellow after autoclaving. Hybridization buffer: Final concentration for 100 ml 50% formamide 50 ml of formamide 5x SSC 25 ml of 20xSSC 1 mg/ml Torula RNA 2 ml of 50 mg/mltorula RNA 100 µg/ml heparin 10 mg heparin 1x Denhart s 1 ml 100xDenhart s 0.1% Tween 20 (Sigma P-1379) 0.1 ml Tween % CHAPS (Sigma C3023) 0.1 gm CHAPS 10 mm EDTA 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 MgCl 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
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