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RNA Whole Mount In Situ Hybridization

RNA Whole Mount In Situ Hybridization

Core protocol for both mouse and chick embryos Cepko/Tabin lab

Embryo preparation

  1. Fix embryos in fresh 4% paraformaldehyde (or a thawed frozen aliquot) in PBS at 4° C overnight.
  2. Dissect embryos (as needed) into PBT, and wash once in PBT for 5 minutes with gentle rocking.
  3. Dehydrate embryos into methanol using a graded methanol/PBT series (25%, 50%, 75%, 100% methanol). Allow embryos to rock gently at room temperature for 5 minutes in each wash solution.
  4. Do one additional wash in 100% methanol (5 minutes). At this point, embryos can be stored for up to 1 month (or more) at -20°C (use either glass scintillation vials or plastic screw-cap tubes).

Day 1: Prehybridization and Hybridization

  1. Rehydrate samples in a 75%, 50%, 25% methanol/PBT series. Wash twice in PBT for 5 minutes at room temperature.
  2. leach embryos (if necessary) with 6% hydrogen peroxide in PBT for 1 hour at room temperature with gentle rocking. (Note: bleaching time can be reduced or extended if desired.) Embryos must have been previously dehydrated in methanol if you are going to include the bleaching step!!
  3. Wash 3 times with PBT for 5 minutes each.
  4. Proteinase K treatment:
    Chick embryos: For younger embryos (< st 10), treat with 1 to 3 g/ml proteinase K in PBT for 15 minutes at room temperature. A similar treatment can be used for detecting ectodermal gene expression. For older embryos, (> st 10) a harsher treatment with proteinase K is necessary. Two parameters can be manipulated: the concentration of proteinase K and/or the length of treatment. Embryos younger than stage 18 are usually treated with 10 mg/ml proteinase K for about 15 minutes at room temperature. For embryos between stages 18 and 24, proteinase K treatment (10 mg/ml) can be extended for 20 to 25 minutes at room temperature. For stages 26 to 29, enzyme treatment with 10 mg/ml proteinase K can last for up to 40 minutes at room temperature. Alternatively, for embryos at stage 26 and older, the concentration of proteinase K can be increased while keeping the incubation time (15 minutes at room temperature) constant:
    stage: concentration of proteinase K
    26-27: 20 mg/ml
    28-29: 30 mg/ml
    30-31: 40 mg/ml
    32 and older: 50 mg/ml
    Mouse embryos:incubate in 10 mg/ml proteinase K in PBT for 5-10 minutes, depending on embryo stage. Use the following information as a guideline:
    embryo age: duration of incubation
    6.5 dpc: 4 min
    7.5 dpc: 4-5 min
    8.5 dpc: 6 min
    9.5 dpc: 10 min
    10.5 dpc: 15 min
  5. Wash 10 minutes in 2 mg/ml glycine in PBT (make fresh).
  6. Wash two times for 5 minutes each with PBT.
  7. Postfix with 4% paraformaldehyde and 0.2% glutaraldehyde (0.2 ml of 25% stock per 25 ml) in PBT for 20 minutes at room temperature.
  8. Wash 2 times for 5 minutes each in PBT.
    For mouse embryos, a slow equilibration in hybridization solution is suggested. This approach has not been routinely used for chick embryos, but it could probably be used successfully.
  9. Wash 10 minutes in a 1:1 mixture of hybridization solution/PBT
  10. Wash 10 minutes in hybridization solution.
  11. Incubate at 70°C in hybridization solution for at least 1 hour.
  12. Replace hybridization solution with fresh, add RNA probe (typically anywhere from one-fiftieth to one-tenth of a transcription reaction) and incubate overnight at 70° C.

Day 2: Post-hybridization washes, blocking, and antibody incubation

  1. Prewarm solution I to 70°C. Wash embryos 3 times for 30 minutes each at 70° C with prewarmed solution I .
  2. Prewarm solution III to 65°C. Wash embryos 3 times for 30 minutes each at 65°C with prewarmed solution III.
  3. Wash 3 times with fresh TBST for 5 minutes each at room temperature.
  4. Blocking step:
    For chick embryos, block with 10% heat-inactivated sheep serum in TBST for at least one hour at room temperature.
    For mouse embryos, block embryos by incubating at room temperature for 60-90 minutes in blocking solution (10% heat-inactivated sheep serum and 0.1% Boerhinger Mannheim blocking reagent in TBST).
  5. Preabsorb the antibody, if desired. (Note: A number of people do not preabsorb the antibody for chick embryos and still get good results, using a dilution of 1:5000 to 1:10,000).
    For chick embryos, put roughly 3 mg of chick embryo powder into a microtube with 0.5 ml TBST. Heat at 70° C for 30 minutes, vortex for 10 minutes. Cool on ice, add ml sheep serum and 1 m l anti-dig AP antibody. Shake gently at 4° C for 1 hr. Spin in microfuge for 10 minutes at 4° C. Collect supernatant into a 15 ml tube. Dilute with 1% sheep serum/TBST to a volume of 2 to 8 mls per vial of embryos, depending on the desired antibody dilution.
    For mouse embryos, follow the above procedure but use roughly 3 mg of mouse embryo powder and 0.5 ml TBST plus Boerhinger blocking reagent.

  6. Remove blocking solution from embryos. Add antibody and incubate overnight at 4°C.

Day 3: Post-antibody washes

  1. Wash 3 times for 5 minutes each with TBST at room temperature.
  2. Wash 5 times for 1 to 1.5 hours in TBST at room temperature.
  3. Wash overnight in TBST at 4°C with gentle rocking.

Day 4: Color development

  1. Wash 3 times in NTMT for at least 10 minutes each at room temperature.
  2. Remove NTMT, add reaction mix (125 mg/ml BCIP and 250 m g/ml NBT in NTMT) and allow the reactions to develop at room temperature with gentle rocking. Throughout the development reaction, keep samples covered with aluminum foil to protect them from light and check them periodically to monitor the progress of the reaction.
  3. When the reaction is judged complete, wash with PBS or PBT and post-fix in 4% paraformaledehye/ 0.1% glutaraldehyde.
  4. Wash two to three times in PBS.


All solutions should be made with high-quality reagent grade water that is RNase-free. (Note: We routinely use water directly from our purification system without DEPC treatment.) In addition, all solutions containing Tween-20 should be filtered and kept sterile.

10X PBS (1 liter):

80 g NaCl
2 g KCl
14.4 g Na2PO4
2.4 g KH2PO4
(adjust to pH 7.4 with HCl and bring to 1 liter with water)


1X PBS plus 0.1% or 1%* Tween
*NOTE: For mouse embryos, it is recommended that all solutions with Tween contain 1%, rather than 0.1%.
Tween is routinely used at 0.1% in the chick protocol.

Hybridization solution:

50% formamide
5X SSC, pH 4.5 (use citric acid to pH)
1% SDS
50 mg/ml yeast tRNA
50 mg/ml heparin

Solution I:

50% formamide
5X SSC, pH 4.5
1% SDS

Solution III:

50% formamide
2X SSC, pH 4.5
0.1% Tween (NOTE: this is only included for
young chick embryos, approx. st 4-st 9)

Sheep serum:

inactivate by heating to 70°C for 30 minutes, store in aliquots at -20°C.

10X TBS (for 1 liter):

80 g NaCl
2 g KCl
250 ml 1M Tris-HCl, pH 7.5
(bring to 1 liter with water)


1X TBS plus 0.1/1% Tween
2 mM (0.5 mg/ml) Levamisole
(NOTE: most people no longer add levamisole to TBST)


100 mM NaCl
100 mM Tris-HCl, pH 9.5
50 mM MgCl2
0.1/1% Tween-20
2 mM (0.5 mg/ml) Levamisole

200X NBT:

50 mg/ml NBT in 70% DMF

200X BCIP:

25 mg/ml BCIP in water