The Zebrafish Science Monitor, Vol 3 (6)
WHOLE-MOUNT IN SITU HYBRIDIZATION OF THICK TISSUE SECTIONS
By Qiling Xu and David Wilkinson; Randall Institute, King's College London, 26-29 Drury Lane, London WC2B 5RL, UNITED KINGDOM
This is a method involving RNA in situ hybridization of unmounted tissue sections by a whole-mount procedure. It is suitable for large embryos, but may be difficult to carry out with small pieces of tissue that can easily be damaged or lost during the washes. It provides more sensitivity for detection of RNAs than obtained using thin tissue sections mounted on slides, and avoids the penetration problems with whole-mounts of large embryos.
1. Fix embryos in 4% paraformaldehyde in PBS overnight at 4 C.
2. Rinse in PBS, then process for sectioning as described below. We have used embryos embedded in paraffin wax, but it is likely that cryostat sections work at least equally well. Cut ~50m sections.
3. If wax sections have been cut, dewax with Histoclear 3 times for 5 min, then wash in 100% methanol 3 times for 5 min, and go to step 5.
4. If cryostat sections have been cut, dehydrate in 5 min washes of 25%, 50%, 75% methanol in PBS, then twice in 100% methanol.
5. Rehydrate in 5 min washes of 75%, 50%, 25% methanol in PBS and 3 times in PBT.
6. Treat with 10g/ml proteinase K in PBT for 10-15 min at room temperature.
7. Rinse briefly with PBT and then refix with 4% paraformaldehyde in PBS for 20 min.
8. Wash 4 times for 5 min each with PBT.
9. Prehybridize in hyb mix at 65 C for 2-3 h.
10. Replace with hyb mix containing DIG-labelled probe and hybridize overnight at 65 C.
11. Wash for 10 min each at 65 C in:
Solution 1
3:1 solution 1: 2 x SSC
1:1 solution 1: 2 x SSC
1:3 solution 1: 2 x SSC
2 x SSC
12. Wash twice for 30 min each at 65 C in 0.2 x SSC
13. Wash for 10 min each at room temp in:
3:1 0.2 x SSC: PBT
1:1 0.2 x SSC: PBT
1:3 0.2 x SSC: PBT
PBT
14. Block with blocking solution for 60 min.
15. Replace with 1/5000 diluted AP-coupled anti-DIG antibody in blocking solution and incubate at 4 C overnight.
16. Wash in TBT 6 times for 20 min each.
17. Equilibrate with NTMT, 3 times for 5 mins.
18. Stain with NBT/BCIP in NTMT.
19. After appropriate amount of staining has been obtained, wash three times with PBT.
20. Equilibrate in 70% glycerol in PBT and mount under a coverslip. The sections may appear wrinkly, but by placing them on a slide in a minimum amount of liquid and gently unfolding any creases, they can be nicely flattened under a coverslip (use silicon grease to support the edges of the coverslip).
Solutions
PBT: PBS, 0.1% Triton X-100
Hyb mix: 50% formamide, 5 x SSC, pH 6.0, 0.5 mg/ml yeast RNA, 0.1% Triton X-100, 50 g/ml heparin (This is the hyb mix we have used, but your favorite hyb mix for whole-mounts should also work)
Solution 1: 50% formamide, 5 x SSC
Blocking solution: 2 mg/ml BSA, 5% sheep serum, 1% DMSO in PBT
TBT: 50 mM TrisHCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100.
NTMT: 100mM NaCl, 100mM TrisHCl pH 9.5, 50mM MgCl2, 0.1% Triton X-100. Make from concentrated stocks on day of use (because the pH will decrease during storage due to the absorption of carbon dioxide).
NBT: 75 mg/ml in 70% dimethylformamide (store at -20 C); use 4.5l per ml NTMT.
BCIP: 50 mg/ml in dimethylformamide (store at -20 C); use 3.5l per ml NTMT.
Preparation of wax sections
1. After fixation and washing in PBS, dehydrate by washing for 30 min each in 25%, 50%, 75% methanol in PBS, 3 times in 100% methanol, then 2 times in Histoclear.
2. Incubate for 20 min at 60 C in 1:1 Histoclear: paraffin wax, then 3 times in paraffin wax.
3. Transfer to suitable mould, orientate as required and allow wax to set.
4. Cut 50m sections on a microtome. Continue the hybridization protocol at step 3.
Preparation of cryostat sections
1. After fixation and washing in PBS, embed in 5% sucrose, 1.5% LMP agarose (Sigma) and then leave the blocks in 30% sucrose, 1% paraformaldehyde overnight at 4 C to equilibrate.
2. Cut 50m thick sections on a cryostat, incubate in 4% paraformaldehyde in PBS for 20 min, then continue hybridization protocol at step 4.
The Zebrafish Science Monitor, Vol 3 (6)
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