RE: Seeking H&E stains procedure for Ground Plastic section

From:"Smith, Allen"
40 to 100 microns is awfully thick for plastic sections.  40 to 100 nanometers (i.e., millimicrons) is customary.
I have had fair results staining 100 to 500 nanometer plastic sections as follows:
1. Cover sections on glass slide with a few drops of Harris hematoxylin.
2. Heat on 70 degrees C hotplate, 2 minutes, adding a drop of hematoxylin every 15 seconds.
3. Remove slide from hotplate and allow to cool.
4. Rinse with distilled water.
5. Set slide in distilled water, 5 min.
6. Set slide in tap water, 5 min.
7. Wipe underside of slide dry.
8. Heat on hotplate, 5 min.
9. Cool slide.
10. Cover sections with a few drops of aqueous 1% eosin Y.
11. Heat on hotplate 1 min, adding a drop of aqueous eosin every 15 sec.
12. Cool slide.
13. Rinse in 80% ethanol, 5 seconds.
14. Wipe underside of slide dry.
15. Heat on hotplate, 5 min.
16. Cool slide.
17. Examine sections as is or coverslip with Fisher's "Permount".

Allen A. Smith, Ph.D.
Barry University
School of Graduate Medical Sciences
    Podiatric Medicine and Surgery
Miami Shores, Florida  33161-6695

-----Original Message-----
From: Bernard Ian R SSgt 59 CRES/MSROP [mailto:Ian.Bernard@LACKLAND.AF.MIL]
Sent: Sunday, January 05, 2003 1:37 PM
To: Histonet (E-mail)
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
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