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

From:Bryan Llewellyn

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 -----
From: Bernard Ian R SSgt 59 CRES/MSROP
To: Histonet (E-mail)
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

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