Re: [Histonet] Re: H&E Staining for SPI-Chem Low Acid GMA

From:"Gayle Callis"

We never had much plastic background staining with Gill 3 hematoxylin on 
thin GMA tissue sections e.g. 1 to 3 um, and the staining time was never 30 
minutes.  Even a thicker GMA section was stained no more than 10 minutes, 
rinsed and blued with Scotts tap water, air dried then stained with eosin 
phloxine. We never had to differentiate our hematoxylin with any kind of 
acid HCL or acetic acid clarifier.   Your section will probably be totally 
stained within 5 minutes, even with GMA media.  We never used clarifier but 
the slight blue imparted to the plastic never was an  factor for viewing or 
photography.   If your section is in the very thin thickness range, 30 
minutes staining time is NOT going to make the nuclei bluer as there is just 
not enough tissue there to stain in the first place. At that thickness you 
are cutting through the nuclei.  The nuclei look more like nuclei in EPON 
sections cut very thin (0.5 to 1 um thick).  HOwever, the longer the PLASTIC 
is in the stain, the more likely this will pick the hematoxylin and cause 
unsightly background.

We found eosin phloxine gave better tinctorial quality to the sections than 
just eosin y.

Gayle M. Callis'

----- Original Message ----- 
From: "Hobbs, Carl" 
Sent: Wednesday, August 27, 2008 8:14 AM
Subject: [Histonet] Re: H&E Staining for SPI-Chem Low Acid GMA

Hi Scott,
GMA resin will take up will need to differentiate the 
Gills in acid.
After Gill's staining:
1. Rinse in gently running tap water until clear.
2. Give slide say, 5 dips, in 0.5% HCL in distilled water, wash in tapwater 
5mins ( I assume that your tapwater is slightly alkali - if not, then follow 
with Scott's) Examine microscopically. You will thus see if more 
differentiation in HCL is reqd or not.
3. Once you have optimally stained nuclei, counterstain in Eosin.
4. Wash in gently running tap water until excess eosin is removed and you 
are happy with balance ( this is slow, relative to alcohol, with no danger 
of sections becoming wrinkled because of the alcohol softening it.)
5. Carefully/firmly blot sections dry. I now leave them in a 60C oven ( only 
cos that's all I have) for 60 mins, minimum.
6. Place into xylene. Leave for 10 mins before mounting .
A nice easy, reliable method, imho.
NB: after cutting/mounting sections, leave in 60C oven for 2 hrs to ensure 
that they are "baked" on.

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