[Histonet] More on Re: IHC in resin embedded sections

[Gayle Callis]

There is a way to remove resin, but it is not very friendly at times to the 
tissue, called sodium ethoxide.  Recipe for this can be found in Histonet 
archives, www.histosearch.org as it was discussed not too long ago.  You 
can search for this info IF you think you need it.

Also, antigen retrieval methods have been done on tissues embedded in 
resins for EM purposes.    Shi and Taylors book, Antigen Retrieval 
Techniques has a whole chapter devoted to this.  The book can be purchased 
from Eaton Publishing, and is worth having around a lab for other retrieval 
technics, hints, etc.   I have seen retrieval methods published for EM 
resins, but you would have to hit PUBMED to find them, they can be found in 
EM type journals - it is something we do not do, but cruising journals can 
be fun.

An aside -  have heard Spurrs is being discontinued as one of the 
ingredients is not being made anymore.



At 06:10 PM 1/31/2005, you wrote:
>    Your success will depend on the antigen in question and how it reacts 
> to fixation and embedding.
>I would try fixing in buffered formalin or paraformaldehyde and embedding 
>in Araldite or an Epon substitute, avoid Spurr's resin. There are some 
>resins that are quite antigen friendly, Lowicryl for one, but they are a 
>bit harder to use. You might be able to use some glutaraldehyde in the 
>fixative but I would first find a method that works, then try for better 
>morphology with stronger fixation.
>    You might also try Vibratome sections of 40-100 microns before 
> embedding. Then do IHC on those before embedding or just use those 
> sections to check orientation of the tissue.
>    Frozen sections of fixed material should provide very good morphology 
> if freezing is done very quickly after proper cryoprotection.
>
>Geoff
>
>Tessa Murray wrote:
>
>>
>>   Dear Histonetters,
>>
>>   I  am  looking  for  some advice about the feasibility of doing IHC on
>>   tissues embedded for EM work.  I need to section very small tissues in
>>   a  specific orientation which is difficult to determine when embedding
>>   in  paraffin.   I  was  wondering  if we could borrow the EM technique
>>   where  (as  I  understand)  the  tissue  is embedded in resin and then
>>   temporarily  fixed to a "peg" for sectioning - thus allowing us to cut
>>   some  sections,  check  the  orientation  and  then  rotate  the block
>>   accordingly  until  we get the orientation exactly right.  Obviously I
>>   am  concerned  about  how  well  antibodies are going to recognize the
>>   tissue  after  processing, how are the sections de-resined, is there a
>>   specific  antigen  retreival  step  for  resin  tissues etc.  Any help
>>   appreciated  -  perhaps  there is a way to adjust sectioning angles in
>>   FFPE  tissues  that  I've  not  thought of?  Frozen tissues are not an
>>   option  as we want to preserve morphology as much as possible.  Cheers
>>   guys.
>>
>>   Tessa J Murray PhD
>>   Tufts University School of Medicine
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>
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>--
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>Geoff McAuliffe, Ph.D.
>Neuroscience and Cell Biology
>Robert Wood Johnson Medical School
>675 Hoes Lane, Piscataway, NJ 08854
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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)