Imaginal Disc In Situ

Use clean tubes and pipette tips that are RNase free. Water is directly from Millipore distilled water machine (18.0 ohms or higher).

PBT is clean PBS + 0.1% tween.

  1. Fix imaginal discs in 4% PFA (in 1x PBS) for 20 min.
  2. Wash with PBT 2 times briefly.
  3. Wash with 100% methanol for 5 min.
  4. Wash with 75% methanol (25% PBT) for 5 min.
  5. Wash with 50%, 25% methanol for 5 min.
  6. Wash with PBT 3x for 5 min. each. (Some say that proteinase K step is dispensible, but I have not explored that possibility myself).
  7. Treat with proteinase K (10ug/ml) for 5 min.
  8. Wash briefly with PBT twice.
  9. Postfix with 4% PFA for 20 min.
  10. Wash 3x
  11. Acetylation (essential for imaginal disc in situ) : to make solution, combine 9.25g triethanolamine HCl Sigma Cat #T1502 and 1.12ml 10N NaOH in 500 ml dH2O. Mix well. Immediately before adding solution to discs, add 12.5ul acetic anhydride into 5ml of Acetylation solution and shake solution vigorously. Treat imaginal discs with this solution for 10 min.
  12. Wash 3x with PBT.
  13. Prehybridize with 100ul HB4 for 1 hour at 65°C. (HB4 ; 50% formamide, 5x SSC, 50ug/ml heparin, 0.1% tween-20, 5mg/ml torula yeast RNA (Sigma).
  14. Make hybridization mix; add 1-2ul of riboprobe to 100ul of HB4 and heat at 80°C for 10 min., and then chill on ice for 5 min.
  15. Add hybridization solution to your specimen and incubate overnight at 65°C.

NEXT DAY

  1. Wash at 65°C: 2x 15 min. in 50 % formamide, 2x SSCT (SSCT : SSC + 0.1% tween-20).
  2. At 65°C : 15 min. in 2x SSCT
  3. At 65°C : 3x 15min. in 0.2X SSCT
  4. Wash in room temperature, 3x 20 min. in PBT (from here, don’t worry about RNase)
  5. Incubate with anti-DIG-AP Ab (Boeringer) at 1:2000 for 2hours.
  6. Rinse with PBT 3x 20 min.
  7. Rinse 2x with AP buffer (0.1M Tris pH 9.5, 50mM MgCl2, 0.1M NaCl, 0.1% tween-20).
  8. Detect with 4.5ul NBT (75mg/ml) and 3.5ul BCIP (50mg/ml) in 1ml AP buffer. It can take anything from 5 min. to a few hours.
  9. Wash with PBT.
  10. Mount on slideglass.