[Histonet] Which Collagen type for IHC in mouse tissue

John Kiernan jkiernan <@t> uwo.ca
Tue Dec 14 12:42:20 CST 2004

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 
   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


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.


Collagen, Collagenase, Over-digestion, Immunohistochemistry 


Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD The
Molecular Biology of the Cell 1989; 808-8152nd ed

Barsky SH, Roa NC, Restrepo C, Liotta LA Immunohistochemical
enhancement of basement membrane antigens by pepsin:
applications in diagnostic pathology Am. J. Clin. Pathol.
1984; 82: 191-194

Barsky SH, Siegal G, Jannotta F, Liotta LA Loss of basement
membrane components by invasive tumors, but not by their
benign counterparts Lab Invest. 1983; 49: 140-148

D'Andrea MR Evidence linking autoimmunity to neuronal cell
death in Alzheimer's disease Brain Res. 2003; 982: 19-30

D'Andrea MR, Reiser PA, Gumula NA, Hertzog BM,
Andrade-Gordon P Application of triple-label
immunohistochemistry to characterize inflammation in
Alzheimer's disease brains Biotech. & Histochem. 2001; 76:

D'Andrea MR, Reiser PA, Polkovitch DA, Gumula NA, Belkowski
S, Branchide B, Hertzog BM, Schmidheiser D, Belkowski S,
Gastard M, Andrade-Gordon P The use of formic acid to
embellish amyloid plaque detection in Alzheimer's disease
tissues misguides key observations Neurosci. Lett. 2003;
342: 114-118

Farquharson C, Robins SP Immunolocalization of collagen
types I and III in the arterial wall of the rat Histochem.
J. 1989; 21: 172-178

Grimaud JA, Druguet M, Emonard H, Peyrol S, Barioz CM,
Guerret S Collagen immunotyping NATO ASI Series, Series A:
Life Sciences 1985; 93: 487-514

Higuchi I, Suehara M, Iwaki H, Nakagawa M, Arimura K, Osame
M Collagen VI deficiency Ullrich's disease Ann. Neurol.
2001; 49: 54

Kallioinen M, Autio-Harmainen H, Dammert K, Risteli J,
Risteli L Basement membrane laminin and type IV collagen in
various benign and malignant adnexal tumors of the skin: An
immunohistochemical study J. Invest. Derm. 1984; 83: 276-280

Kasper M, Schuh D, Muller M Immunochemical localization of
the ß subunit of prolyl 4-hydroxylase in human alveolar
epithelial cells Acta Histochem. 1994; 96: 309-313

Liotta LA Tumor invasion and metastasis-role of the
extracellular matrix: Rhoads Memorial Award Lecture Cancer
Res. 1986; 46: 1-7

Lowry A, Wilcox D, Masson EA, Williams PE
Immunohistochemical methods for semiquantitative analysis of
collagen content in human peripheral nerve J. Anat. 1997;
191: 367-374

Rucklidge GJ, Dean V Immunochemical techniques in the study
of protein degradation with particular reference to collagen
Meth. Surv. Biochem. Anal. 1987; 17: 387-392

Sano J, Ishida A, Ishizaki M, Sugisaki Y, Yamanaka N, Masugi
Y Immunofluorescent visualization of type IV collagen in the
glomerular capillary basement membrane after digestion with
a microbial protease Biomed. Res. 1984; 5: 387-392

Shi S-R, Cote RJ, Taylor CR Antigen retrieval
immunohistochemistry: past, present, and future J.
Histochem. Cytochem. 1997; 54: 327-343

Taylor CR, Shi S-R, Chaiwun B, Young L, Imam SA, Cote RJ
Strategies for improving the immunohistochemical staining of
various intranuclear prognostic markers in formalin-paraffin
sections: androgen receptor, estrogen receptor, progesterone
receptor, p53 protein, proliferating cell nuclear antigen,
and Ki-67 antigen revealed by antigen retrieval technique
Human Pathol. 1994; 25: 263-270

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John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
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 <@t> excaliburpathology.com
> >www.excaliburpathology.com
> Gayle Callis
> 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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