Bernard,
I never worked with plastic sections at 40-100 micrometres as
you are. I used a butyl/methyl methacrylate mixture and cut one
micrometre renal biopsies, so the protocol below may or may not be useful to
you. It worked quite well for what I was doing.
1. Remove plastic with acetone for one hour
at RT. This may not be feasable for 40 micron sections but is worth a
try.
2. Wash with water to ensure the sections
are wetted.
3. Place in preheated Mayer's or Cole's
hemalum for an hour or longer. Change the time to get the desired
depth of staining.
4. Wash with water and blue. I used
0.5% aqueous sodium acetate.
5. Wash with water.
6. Counterstain with eosin Y ws.
Adjust temperature, time and concentration to get what you want.
7. Rinse with water to remove excess
etc.
8. Dehydrate, clear and mount as
usual.
Both Mayer's (Langeron's) and Cole's hemalums are
progressive, so require no differentiation. Mayer's is more selective for
nuclei, Cole's staining is similar to a differentiated regressive stain.
Formulae are on StainsFile.
Bryan Llewellyn
----- Original Message -----
Sent: Sunday, January 05, 2003 10:37
AM
Subject: Seeking H&E stains procedure
for Ground Plastic section
Hello again, to all fellow,
Histo Plastic Researchers,
My Pathologist is adamant in me, finding a H&E procedure
that will work, to stain some plastic processed (Methyl Methacrylate) pig
& rabbit eye implants. I mentioned to him about other stains out
there, that work similarly to the H&E and stains
better. However, he insists that the H&E will give him a
familiar and better contrast.
The one method I have, worked
(acceptable to him) for another plastic procedure( A Butyl Methyl
Methacrylate), but not this one. The stain came out to light.
I even restained the slides following the same procedure again. And it
still turned out light.
My adopted Plastic
H&E Method abbreviated to key reagents:
1. I surface etched the
plastic slides before staining to allow for stain penetration in 1 % Formic
Acid ( 2 mins), 50/50 ETOH and rinse in Distilled H20.
2. Stain 6 min in Harris
Hematoxylin
3. Differentiate in 0.5 %
Acid Alcohol ( HCL in 70 % Alcohol)
4. Blue in Ammonia
water (12 drops Ammonium Hydroxide in 250 mls Distilled
water)
5. Stain 1 min in Eosin Y
solution
6. Dehydrate to Xylene
substitute.
These sections are less than
100 microns thick, no less than 40 microns
I need help with a good
procedure.
Thanks in
advance