H&E on plastic, which plastic? Re: [Histonet] eosin hematoxylin plastic stain

[Gayle Callis]

Myriam,

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 
bone sections.




4, you wrote:
>hi
>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
>myriam
>natural implant
>france
>
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)