Swidbert R Ott wrote:
>> 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 love to help! my first question is why 30 microns? next when I
do frozens I've dried them under dessicant conditions for 1hr followed by
storage in a freezer with dessicant in the slide box. DO you post-fix
with someting like ethanol or acetone? The effects you describe may
relate the the so-called edge effect that I belive is thought to occur
'cause those areas are less-than well rinsed due to physics of washing
and etc. What tissue are you using? Are these 30 micron sections
typical for this tissue?
markH
