Re: [Histonet] Which Collagen type for IHC in mouse tissue

From:John Kiernan

A recent (2004) publication by M.R.D'Andrea describes 
another way to unmask collagen for immunohistochemistry. 
Summary follows. The paper also includes some
interesting information about collagenase.
______________________________

   Publisher:   Taylor & Francis Health Sciences, part of
the Taylor & Francis Group 
   http://journalsonline.tandf.co.uk 
   Journal:  Biotechnic & Histochemistry
   Issue:   Volume 79, Number 2 / April 2004  
   Pages:   55 - 64  
   URL:   Linking Options  
   DOI:   10.1080/10520290410001728972  

Collagenase predigestion on paraffin sections enhances
collagen immunohistochemical detection without distorting
tissue morphology
Biotechnic & Histochemistry  79 (2) 55-64

by  MR D'Andrea 

Johnson & Johnson Pharmaceutical Research and Development
Drug Discovery Pennsylvania 19477-0776 Spring House

Abstract: 

Reliable immunohistochemical detection of collagen in
formalin fixed, paraffin embedded tissues requires protease
digestion. While these pan-proteases (pepsin, trypsin,
protease K, etc.) enhance collagen detection, they also
digest many other tissue proteins and produce poor cellular
morphology and unrecognizable cellular structures. Balancing
the conditions (protease type, concentration, incubation
time and temperature) to digest some, but not all, proteins
in a tissue section while optimizing collagen detection
requires one to compromise improved collagen immunolabeling
with adequate cellular morphology. Furthermore, optimal
conditions for digesting tissue proteins to enhance collagen
detection vary among tissue types and their fixation.
Although brain is not typically subject to these deleterious
consequences, structures such as epithelium, spermatids,
stroma etc. and other tissues with complicated histology are
profoundly affected. To resolve this technical dilemma, we
discovered a novel use for collagenase to enhance collagen
immunodetection without affecting the noncollagen proteins,
thereby preserving tissue morphology. Collagenase, which is
typically used in vitro for disassociation of cells, has
never been used reliably on formalin fixed, paraffin
embedded tissue sections. This new use of collagenase for
immunohistochemistry promotes increased collagen
immunolabeling, is easy to use, is versatile, and allows
preservation of tissue structure that provides maximal and
accurate histological information.

Keywords: 

Collagen, Collagenase, Over-digestion, Immunohistochemistry 

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_______________

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-- 
-------------------------------
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
   kiernan[AT]uwo.ca
   http://publish.uwo.ca/~jkiernan/
   http://instruct.uwo.ca/anatomy/530/index.htm
_______________________________
Gayle Callis wrote:
> 
> Which collagen do you need, Chemicon has Types 1 through IX, but Type IV
> polyclonal is the only one that they say cross reacts to mouse collagen.
> 
> At 10:34 AM 12/14/2004, you wrote:
> >Have you tried just a histochemical stain for collagen such as Masson's
> >Trichrome?
> >
> >
> >Paula Pierce, HTL(ASCP)HT
> >
> >Excalibur Pathology, Inc.
> >631 N. Broadway
> >Moore, OK 73160
> >405-759-3953
> >contact@excaliburpathology.com
> >www.excaliburpathology.com
> 
> 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)
> 
> _______________________________________________
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