In situ hybridization, brief version (Harland lab)

Brief version of the Harland protocol [1]

Embryos

Fix in 4% formaldehyde, 0.1M MOPS, pH7.4, 2 mM EGTA, 1 mM MgSO4 (MEMFA) for 30 to 60 minutes at room temperature, rinsed with TBST, stored at -20degC in 100% methanol.

Embryo pretreatment

-rehydrate slowly with TBS -rinse twice (5' each) in 0.1M triethanolamine pH 7 to 8 -add 12.5 ul acetic anhydride, mix at room temp for 5'. -add another 12.5 ul acetic anhydride, mix at room temp for 5'. -wash 2X with TBST (5')

Hybridization

-remove most of TBST, gently add 250 ul of hyb buffer, let tube sit on bench until embryos settle down through the more dense hyb solution -remove most of solution, replace with 500 ul of hyb buffer, incubate at 60 degC for as long as possible with mixing (1 hour to overnight) -remove solution, replace with 500 ul of hyb solution containing 1ug/ml DIG labelled antisense RNA probe. Incubate at 60 degC o/n with mixing.

Antibody DIG detection

-remove probe and store at -20C (reusable!!!), replace with 500 ul 50% hyb buffer/50% TBST at 60 degC, mix 5' -remove solution, replace with 60 degC TBST, mix 5', repeat -replace solution with 0.2 X SSC/0.05% tween 20 and wash at 60 deg C for 10', repeat -replace 0.2 X SSC/0.05% tween, cool to 37 degC -add RNaseA to 40 ug/ml, mix at 37 degC for 30' -wash 3 X 30' (or more) at 65 degC in 0.2 X SSC/0.05% tween20 -wash in TBST room temp 2 x 5' -replace TBST with antibody blocking solution, 500 ul, room temp 15' -add 0.25 ul Boehringer AP or HRP-coupled anti-digoxygenin antibody, mix 4 degC o/n -wash in several changes of TBST at room temp (5 hours) -develop using appropriated substrate