Whole-mount in situ hybridization of vertebrate embryos

(version 1.0;    01/27/04)

 

Based on protocols developed by D. Wilkinson (see In Situ Hybridisation: A Practical Approach, ed. D. Wilkinson, IRL Press, Oxford University, 1992), and modified by the labs of A. McMahon, M. Bronner-Frasier, and D. Kingsley.

 

Notes before starting:

 

Fixation: Fix embryos overnight at 4°C in 4% paraformaldehyde (in PBS) and store in 100% methanol at ­20°C. Embryos can be stored for at least several months and still yield good results.

 

RNases: RNases can reduce or eliminate RNA in your embryos and obliterate your riboprobe, so take special care to keep all materials RNase free. New plasticware and sterilized glassware should be used whenever possible. All reagents in the hybridization solution and post-hyb washes should be RNase free (see end of protocol for list of solutions and reagents). Wipe down pipettors and benchtops with ethanol or RNase remover (RNase Away (Invitrogen) or similar).

 

Wash volumes: Pre-hybridization, post-hyb, and immunohistochemistry steps can be done using small volumes in small vials. Silanized glass vials (scintillation vials or Reactivials), or disposable plastic vials both work well. If plastic is used, always choose vials/tubes with screw tops. Nunc cryogenic vials (2-mL, round bottom, internal threads) work very well. All other wash steps should be done with larger volumes. 15-mL or 50-mL conical tubes can be used, but solution changes are sometimes be tricky. Another convenient option is Corning Netwells, which are essentially small strainers that fit into 6-well cell culture plates. Netwells allow easy transfer of embryos from one wash to the next without risk of pouring out embryos with the wash solution. Wash volumes using this method will be approximately 10 mL All steps (except pre-hyb, hybridization, and post-hyb washes) should be done on a rocker, if possible; rocking is essential where noted.

 

DAY 1: PRE-TREATMENT AND HYBRIDIZATION:

 

1.              Rehydrate embryos through a MeOH/PBT series (75%, 50%, 25% MeOH, 5 min each, then PBT 2 X 5 min).

 

2.              Bleach with 6% H2O2 in PBT for 1 h at RT, rocking.

a.     Use 1 mL 30% H2O2 per 4 mL PBT

b.     Extend bleaching step up to 3 hours for heavily pigmented specimens (e.g., older stickleback fry

 

3.              Wash with PBT 3 X 5 min.

 

4.              Treat with 10 µg/mL Proteinase K in PBT at RT, rocking.

a.     The activity of different batches of PK may differ significantly, so you may need to calibrate your batch.

b.     This treatment will digest superficial tissue layers, and eventually the entire embryo, if allowed to proceed too long.  Some suggesting starting points for superficial targets (e.g., hind limb buds) in sticklebacks (times for deeper targets should be extended; stages from Swarup, 1959):

                                                     i.     Embryos: 2-5 min

                                                      ii.     Up to stage 29: 10-15 min

                                                        iii.     Stage 30+: 20 min

c.     Suggested times for chick embryos (stages from Hamburger and Hamilton, 1951):

                                                     i.     AER markers (any stage): 0-2 min

                                                      ii.     St 4-6: 2 min

                                                        iii.     St 8: 5 min

                                                       iv.     St 10-13: 10 min

                                                      v.     St 13-16: 15 min

                                                       vi.     St 20: 20-25 min

                                                         vii.     St 25: 30-40 min

 

5.              Wash in 2 mg/mL glycine in PBT (make this solution fresh).

 

6.              Wash with PBT 2 X 5 min.

 

7.              Postfix with 4% paraformaldehyde + 0.2% Glutaraldehyde (Sigma G-5882) in PBT.

 

8.              Wash with PBT 2 X 5 min.

 

9.              Transfer embryos to small-volume vials, add 500 µL of pre-hyb solution (hybridization solution without probe, pre-warmed to 70°C), swirl embryos, decant solution, add 1 mL fresh pre-hyb. Incubate in a water bath at 70°C for 1-2 h.

 

10.           Remove pre-hyb, add 250 µL of pre-warmed hybridization solution (with probe) swirl embryos, decant solution, add 750 µL of fresh hybridization solution. Final probe concentration in the hyb solution should be approximately 100 ng/mL. For most probes, a single overnight hybridization should suffice. For weak probes, try hybridizing over two nights.

 

 

DAY 2: POST-HYB WASHES AND ANTIBODY INCUBATION

 

1.              Replace hyb solution, wash with Solution I (pre-warmed to 70°C) 3 X 30 min. Swirl embryos occasionally during washes. During the third wash, lower temperature to 65°C.

 

2.              Wash with Solution II (pre-warmed to 65°C) 3 X 30 min. Swirl embryos occasionally during washes.

a.     Sticky embryos: during this and the previous set of washes, small embryos may stick to the sides of the tubes. Usually, this does not indicate a major problem. However, if the embryos are very sticky and begin to accumulate debris on them, then they may have been over-digested with Proteinase K.

b.     RNase: some protocols have an RNase step between the Solution I and II washes to reduce non-specific signal. This step is probably not necessary for in situs with chick and stickleback embryos/fry, but it does appear to help with mouse embryos. RNase treatment (between Solution I and II washes):

                                                     i.     Wash with 0.5 M NaCL, 10 mM Tris pH 7.5, 0.1% Tween-20 (TNT) 3 X 5 min at RT.

                                                      ii.     Treat with 100 µg/mL RNase in TNT, 2 X 30 min at 37°C, rocking.

                                                        iii.     Wash with TNT:Solution II (1:1) 5 min at RT, then proceed to Solution II washes.

 

3.              Wash embryos in MAB buffer with 2 mM Levamisole (MW = 240.8) and 0.1% Tween-20 added that day, 3 X 5 min.

 

4.              Pre-block embryos for 3 h in MABLev,Tw +2.% Roche blocking powder + 10% heat inactivated sheep serum. Warm solution to dissolve proteins; solution will remain cloudy.

a.     Optional: pre-absorb antibody

                                                     i.     Put 3 mg of embryo powder in a screw-top microcentrifuge tube

                                                      ii.     Add 500 µL MABLev,Tw + 2% blocking powder, incubate 30 min at 70°C

                                                        iii.     Vortex for 5 min

                                                       iv.     Cool on ice, add 5 µL of sheep serum and 1 µL of anti-DIG-AP antibody

                                                      v.     Shake gently at 4°C for 1 h

                                                       vi.     Spin tube at 4°C for 10 min

                                                         vii.     Collect supernatant in 15 mL conical tube

                                                          viii.     Dilute supe to 2 mL with MABLev,Tw + 2% blocking powder + 1% sheep serum

 

5.              Incubate embryos in anti-DIG-AP antibody mix overnight at 4°C.  If antibody was not pre-absorbed, dilite 1:2000 in MABLev,Tw + 2% blocking powder + 1% sheep serum. Note: this solution can be re-used, and will likely give more specific staining in subsequent experiments.

 

DAY 3: POST-ANTIBODY WASHES

 

1.              Wash in MABLev,Tw 3 X 5 min rocking at RT.

 

2.              Wash in MABLev,Tw 5 X 1-1.5 hr rocking at RT.

 

3.              Wash in MABLev,Tw overnight rocking at 4°C. Note: if background is a problem with larger specimens, washes can proceed for an additional day with minimal loss of specific signal.

 

DAY 4: AP DETECTION

 

1.              Wash in NTMT 3 X 10 min.

 

2.              Replace NTMT with alkaline phosphatase substrate. You mix make your own NBT/BCIP solution, or buy substrates in ready-mixed (Sigma, Roche), kit (Vector Labs), or tablet (Sigma) form.

 

3.              Cover tube or plate containing specimens in foil. Check progress of staining every 20 min to avoid overstaining. For many probes, reactions can continue overnight at 4°C without overstaining.

 

4.              Halt staining reaction

a.     Wash embryos in PBT, 2-3 min. Embryos can still go back into staining solution if staining is weak or incomplete.

b.     Wash in 0.1 M glycine in PBT, 5 min.

c.     Wash in PBT 5 X 1-2 min.

d.     Wash in 100% ethanol 2 X 5 min. Monitor this step closely ­ the staining will become more purple in color, and some background staining will disappear, but remove embryos immediately if specific staining begins to fade.

e.     Wash in PBT 2 X 5 min.

f.      Postfix embryos in 4% paraformaldehyde, at 4°C at least one hour (can leave for several days. Keep embryos wrapped in foil.

 

 

SOLUTIONS AND REAGENTS

 

Use Milli-Q water for all solutions unless otherwise indicated.

 

PBS (1 L)

1.15 g Na2HPO4

8 g NaCl

0.2 g KH2PO4

0.2 g KCl

0.1 g MgCl2 € 6H20

0.133 g CaCl2 € 2H2O

pH to 7.3 and filter sterilize

 

PBT

PBS with 0.1% Tween-20 added. Store up to 1 week at RT.

 

20X SSC (1 L)

175 g NaCl

88.2 g sodium citrate

For use in pre-hyb/hyb solution, use DEPC-treated or nuclease-free water.

pH to 4.5 with citric acid ­ use pH paper rather than pH meter to avoid RNase contamination.

 

 

Pre-hyb and hybridization solution

Composition:                                       For 50 mL:

50% formamide                                   25 mL formamide

5X SSC, pH 4.5                                  12.5 mL 20X SSC, pH 4.5

50 µg/mL yeast tRNA                         125 µL 20 mg/mL tRNA

1% SDS                                              500 mg SDS (or 5 mL 10% SDS)

50 µg/mL heparin                                2.50 mg heparin (or 25 µL 50 µg/µL heparin)

                                                            DEPC-treated or nuclease-free water to 50 mL

 

Sheep serum

Heat inactivate for 30 min at 70°C. Swirl the bottle often so the serum does not coagulate. Store in aliquots at ­20°C.

 

Solution I (post-hyb)

Composition:                                       For 50 mL:

50% formamide                                   25 mL formamide

5X SSC, pH 4.5                                  12.5 mL 20X SSC, pH 4.5

1% SDS                                              5 mL 10% SDS

                                                            7.5 mL DEPC-treated or nuclease free water

 

Solution II (post-hyb)

Composition:                                       For 50 mL:

50% formamide                                   25 mL formamide

2X SSC, pH 4.5                                  5 mL 20X SSC, pH 4.5

0.2% SDS                                           1 mL 10% SDS

                                                            19 mL DEPC-treated or nuclease free water

 

MAB (maleic acid buffer, 1 L)

11.6 g maleic acid

17.4 g NaCl

pH to 7.5 with NaOH ­ use dilute NaOH as pH approaches 7.0 to avoid overshooting.

Add 2 mM Levamisole and 0.1% Tween-20 on day of use and filter.

 

NTMT

Composition:                                       For 100 mL:

100 mM NaCl                                     2 mL 5M NaCl

100 mM Tris, pH 9.5                          10 mL 1 M Tris, pH 9.5

50 mM MgCl2                                     5 mL 1M MgCl2

0.1% Tween-20                                   0.1 mL Tween-20

2 mM Levamisole                                0.048 g Levamisole

                                                            88 mL H2O