H&E on plastic, which plastic? Re: [Histonet] eosin hematoxylin plastic stain
You did not say which plastic you are working with? I presume bone is
undecalcified and embedded in methylmethacrylate? H&E an
methylmethacrylate cannot be done successfully unless you remove ALL the
plastic. You should read this article on why H &E is not going to work on
bone embedded in methylmethacrylate sections. It also discussed
considerations with Glycol methacrylate. First of all hematoxylin is an
aqueous solution and second the molecule is high molecular
weight. Methylmethacrylate is extremely hydrophobic with GMA only less
so. The aqueous hematoxylin stain will not penetrate to the cells. If you
etch the surface of bone in MMA with formic acid, it only removes the
calcium from the bone, but does not remove plastic.
RW Horobin, Staining plastic sections: a review of problems, explanations
and possible solutions. J of Microscopy 131(2):173-186, August 1983.
I do not know of anyone who is successful with an H&E stain on
undecalcified, methylmethacrylate embedded bone section. IIf I recall,
people who have tried H&E on bone in MMA get "muddy" very poor staining
results. Neil Hand did VERY thin undecalcified bone MMA embedded sections
at 2 um, but he removed ALL plastic before H&E staining. Calcium in bone
stains very blue.
I know one person who developed an H&E "look alike" stain (toluidine blue
with basic fuchsin) on undecalcified methylmethacrylate bone
sections. This provided contrast but not a true tissue-chemical
interaction by eosin as with eosinophils or eosinophilia. There is nice
contrast with bone cells, bone, cartilage, soft tissues, etc. This can be
done on thick sections with surface staining - after formic acid etching of
surface. Very nice stain, and plastic did not have to be removed.
H&E is very successful in decalcified bone sections, not sure about 50 um
thick though, and we do a progressive hematoxylin on decalcified bone but
NOT Mayers, you want to get deeper staining of the bone along with other
soft tissue components. In Bancroft and Stevens 4th Edition, Theory and
Practice of Histological Technique, there are pictures of Mayers
hematoxylin on decalcified bone next to Ehrlichs hematoxylin on an adjacent
section. The Mayers looks washed out, the cartilage stains poorly as
compared to Ehrlichs. We have done Ehrlichs hematoxylin on decalcifed bone
sections but on 5 - 10 um thickness. 50 um sections, if the stain could
penetrate all the way through the undecalcified bone and even with
undecalcified will probably mask a lot of what you want to see.
As far as eosin versus eosin phloxine, we prefer the latter for decalcified
4, you wrote:
>i would like to stain plastic sections about 50 um thikness with eosine/
>hematoxyline stain, wich hematoxyline should i use ? :
>hematoxyline solution harris modified (7g/L) or Hematoxyline mayer's (1g/l)?
>i found some protocols with phloxine B and eosin Y, and others with eosin
>only, do you have any information about these use ?
>thank you very much in advance
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Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
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