In situ hybridization, brief version (Harland lab): Difference between revisions

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

Revision as of 16:45, 11 January 2010

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

For fluorescent in situ hybridization see