Re: Keepin GMA brains on slides

From:Gayle Callis

I used to clean slides with detergent wash, rinsed and followed by actone.
However, this was abandoned in favor of regular uncoated/unwashed Fisher's
Finest slides.  Sounds like you are not getting the section perfectly flat
onto the slide initially and/or you have rinsed the slides with water which
swells the plastic (GMA cannot be removed), and spending too much time in
alcohols which tends to raise the plastic ( plus wrinkling or parched earth
look - see comments below on staining)   

Stain dried slides without any rehydration, go directly to hematoxylin  -
this is one of the joys of GMA, no rehydration (we did Gill III for 10 min,
rinse 1 min, (never used an acid clarifying step with GMA), Scotts tap
water substitute for 1 min, rinse then AIR DRY sections BEFORE going into
Eosin for 1 min, rinse rapidly in 95% alcohol twice.  Air dry and coverslip
without ever letting the section sit in xylene for clearing, this causes
nasty wrinkles in plastic as does excessive alcohol rinses after the eosin.
  Look at a stained plastic section before you coverslip, they are actually
not that bad!  Many of the rules that apply to paraffin section staining
(rehydration, dehydration) can be ignored with plastic!  

As for flat sections, the knife has to be extremely sharp.  We preferred
glass triangular knives, trimmed block face with an old blade, changed to a
new blade for every block for actual sectioning with knives freshly broken
for that days work.  Glass, a supercooled liquid, sitting around dulls, the
glass molecules tend to flow a bit over time, same rules applied to EM,
always break a knife just before use.  Go to a RT waterbath (large staining
dish) some people add ammonium hydroxide, a few drops, to flatten the
section, pick up, drain, and go to a 60C oven for an hour.  

IF the tissues are not infiltrated perfectly, they can also give problems.
1 - 2 mm thick tissue samples for best dehydration, also go all the way
through absolute, then infiltrate in GMA overnight in a refrigerator,
transluscent look and tissues sitting on bottom of vial were a must.  If
tissues are too thick with GMA, you can have infiltration and
polymerization problems.  

I was always under the impression that GMA worked best for thin section
projects, 1 - 3 um.

  At 04:20 PM 1/24/02 +0000, you wrote:
>I have JB-4 embedded brain sections that I am cutting at 4 microns that I 
>need to find a slide that these sections will stay on. It looks like they 
>are nice and flat on the slide,but when stained with H&E the tissue traps 
>eosin under the section and cannot be cleared away. We have tried, charged, 
>Poly-L, and silane slides. Polysciences has been helping me with suggestions 
>and I thought I would also ask the experts out there.
>Nancy Maronto
>MSN Photos is the easiest way to share and print your photos: 
Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367
406 994-4303 (FAX)

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