Q: AB penetration prob. in cryostat sections

Swidbert R Ott sro21 at cam.ac.uk
Thu Apr 2 03:57:33 EST 1998


> 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

I could go down to 20 or 15 microns, but I'm not sure whether this would
make any difference. I am doing serial sections of an insect ganglion (ca.
1x1x0.5 mm) where every single section is crucial, but the less sections
the less work... admittedly, if the staining only works on 5-10 microns
sections, I do still a lot of work for nothing. But it looks rather as if
the staining either works (thru and thru, in some sections) or doesn't
work at all. If section thickness was the problem, I would expect
superficial staining that fades into the depth of the section. It looks,
however, more like that the surface is blocked (air-drying effect?, see
below).

I do NO-induced cGMP immuno, so the material is paraformaldehyde-fixed (2h, RT)

What would the effect of ethanol/acetone postfixation be? Is it supposed
to break up the tissue and improve penetration?

Several people have now confirmed to me that the air-drying "cakes" the
tissue and makes it impenetrable for (some) ABs.

-- 
Swidbert R. OTT
Dept. Zoology, Univ. Cambridge/UK



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