Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocalHi every one,
we try to make confocal IF on ~100 µm diameter colospheres.
The main problem we are facing is permeabilization since colospheres have a
rather dense structures preventing efficient penetration of antibody.
Among the different protocols that we tried the best results so far were
obtained when permeabilization (Triton X100 1%) was performed simultaneously
with fixation (PFA 4% - 3h). Nevertheless the antibodies do not reach the
very center of the colospheres. Methanol or ethanol treatment (+/- acetic
acid) for 10 or 20 minutes following PFA or antibody incubation times as
long as 48 hours dit not solve the problem.
I would be happy to get any advise.
Best regards to all.
Bruno
Locked
