in situ hybridization -s/n ratio problem

student sfreckNOsfSPAM at pacbell.net.invalid
Mon Aug 21 22:47:38 EST 2000


after going through all the online protocols i could find
and some of their references im going to try the following
changes: pepsin digestion at various times, 37C (instead of
proteinase K), with and without post-fix in 4%
paraformaldehyde 5min
i was going to add a biotin block after the post-fix
step:ABC soln 30 min RT, PBS washes, 10% H2O2 in PBS 30 min,
then continue with dehydration steps
i was also going to add formamide to all rinses post-hyb.
(which should decrease the background problem)

if you have other methods of blocking endogenous biotin or
other suggestions to change the protocol i would appreciate
the help




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