Re: [Histonet] specimen size for IHC

From:"Jackie M O'Connor"

CM  Van der Loos is the "Dr. House of Histology".  I believe whatever he 
says unequivocally.
I pulled this from the Jan 06 archives.

[Histonet] RE: optimal thickness for cutting of IHC sections
"C.M. van der Loos" 

Dear  Paul,
I totally agree what you
   wrote  about  IHC:  it's just a surface event.
Long time ago we had to prove that our (prehistoric) attempt of
   quantifying an IHC technique wasn't influenced by tissue thickness
   due  to microtome  abberations.  We did cut (cryostat) sections of
   different thickness and guess what: there was hardly any influence
   on the staining intensity. The only thing we observed was
   that   thicker  sections showed  a  higher  non-specific  background
   staining   than  thinner  sections. See:
  CM  van der Lo   os,   MMH   Marijianowski   and   AE   Becker: 
Quantification  in
   immunohistochemistry.  The  measurement  of the ratio’s between
   collagen   types   I   and   I   II.   Histochem   J  (1994)  26,
   347-354<=    /P> 
Chris  van der   Loos, PhD
Dept. of Pathology
Academic Medical Center
   M 2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The   Nether lands 
Date:  Tue,  3  Jan 2006 13:09:20 -0500 
   "Monfils, Paul"
Subject :  RE:
   [Histonet]  optimal  thickness  for  cutting  of  IHC sections< BR>To: 
I  believe  section  thickness  is less critical for IHC
   because  antibodies  are very  large  molecules that don't
   penetrate  tissue  very well, so regardless of
the thickness   of   the   section,   you   are really   only   staining 
   exposed surface  of  the  tissue,  perhaps to a depth of 2
   microns  or so.  We have verified this by electron
   microscopy.   This  is  quite  different  from standard  histochemical 
procedures  where  a  thicker section results in a
   more  intense stain because the small dye molecules penetrate
   the    tissue  readily   and   stain it   all   the   way   through.

Heather Renko  
Sent by:
05/18/2006 01:45 PM


[Histonet] specimen size for IHC

The optimum size for IHC is 3-4mm thick and 1-2cm square to allow adequate 
penetration in the fixation process.  The Immunochemical Staining 
handbook(3rd Ed, published by Dako) states to cut at 4-6. 
  Heather Renko
  In your  invaluable, but,  no  doubt differing opinions, what  is  the 
optimal section thickness   for formalin fixed paraffin processed 
tissues for  immunostaining, using  DAB  as  chromogen?, there has been 
some  discussion here  whether  it  should be 4,5,6, or  7microns, many 

Richard  Edwards

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