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
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
GMA resin will take up Haemalum......you 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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