Re: [Histonet] IHC in resin embedded sections

From:Geoff McAuliffe

    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.


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
> _______________________________________________
> Histonet mailing list

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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