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