Q: AB penetration prob. in cryostat sections

mark mark.haynes at mail.tju.edu
Wed Apr 1 15:19:26 EST 1998


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



More information about the Immuno mailing list