Dear fellow immunocytochemists (?),
could you help me with the following problem: I'm doing
cGMP-immunocytochemistry on 30 mu cryostat sections, which I mount onto
(warm) slides and air-dry. I have severe problems with what seems to be
antibody penetration. My feeling is that the air-drying somehow "cakes"
or "clogs" the tissue so that the ABs do not penetrate properly (despite
having 0.2% and 0.3% Triton in all wash and incubation buffers,
respectively).
Currently I incubate for 24h at RT or 40h in the fridge, and staining is
still partial/patchy/weak. I do get much more staining in sections that
are half detached from the slides, or that have simply been grossly skewed
and distorted when mounting them on the slide. I know that air bubbles
under sections always give more staining and background, but here the
effect is really dramatic, with sharply stained fibres on the detached
parts of the sections and almost no staining on the properly attached
regions/sections.
I also feel that increasing the incubation times further might not really
make a big difference.
I remember faintly that some people did enzyme preincubation (protease?
collagenase?) to improve AB penetration in frozen sections, but I'm not
sure whether my memory serves me right, nor what the reiceipe might be.
Are there any other tricks to solve the problem? (I could do free-floats,
of course, but that would be a real pain because I need serial sections.)
I would really be glad if somebody could help me with this.
Swidbert
--
Swidbert R. OTT
Dept. Zoology, Univ. Cambridge/UK
