Re: Collagen Stains?? (Also M Gabe's book)

From:"J. A. Kiernan" wrote:
> Can anyone recommend a stain that will differentiate different types of
> fibro-cartilage?  I am trying to differentiate the nucleus pulposis from
> annulus fibrosis in spine sections.  My understanding (which is VERY
> limited!!!) is that one is made of Type I collagen and the other is Type II
> collagen.

Immunostaining for different types of collagen might 
be comparable to using a sledgehammer on a nail for making
this distinction. The annulus fibrosus is robust fibro-cartilage
and the nucleus pulposus is, well, pulpy - not richly endowed
with tough collagen and cartilage. This distinction is apparent
in children, but becomes less pronounced in adulthood, when
fibrocartilage invades the territory of the nucleus pulposus.
Weakening of the annulus fibrosus is said to be the cause of
herniation of material from the centre of a disk. (Source:
Gray's Anatomy 38th edn, 1995, which gives several references
for intervertebral disk histology.)

The easiest way to show cartilage matrix, collagen fibres and
cells in the same section is to stain first with alcian blue
at pH 1 (cartilage matrix turqouise-blue) and then with Weigert's 
iron-haematoxylin (nuclei black) and van Gieson (collagen red,
cytoplasm yellow). This sequence requires very little human
judgement to get a good result. The only iffy step is the
nuclear stain. If it's too strong cytoplasms will be greyish
yellow, and if it's too weak the nuclei will be a displeasing
grey or brown colour. Anyone who can do van Gieson can use it
to counterstain after alcian blue.

Now for M. Gabe.

In his big fat book "Histological Techniques" (1976) Manfred
Gabe extolled iron haematoxylin-van Gieson as the best general
staining method ever invented. (Gabe, a distinguished French 
zoologist who did all his lab work without the help of a 
technician, despised H & E and did not include instructions in 
his monumental text. He approved of alum-haematein and eosin
separately, in conjunction with other stains, but he said
the combination of H and E amounted to applying pink paint to
a section with blue nuclei. He wasn't an afficionado of the
subtle shades of orange-pink and bluish-pink!) 

The technical instructions in Gabe's "Histological Techniques"
are very detailed and reflect the man's own experiences with
doing staining methods that he categorizes as "easy" (such as
the ones above or his own one-step trichrome), "difficult" 
(such as trichromes with 2 or more steps; his AZAN procedure
works magnificently if you do as he says) and "fickle" [I think
he used some other word] for methods that can go wrong even if
you proceed according to precedent and protocol (notably silver
and gold stains, especially the ones for central neuroglia
and peripheral axons).

Gabe died a year before the publication of the English 
translation of a revised version his big techniques book,
with the unfortunate consequence of a hopelessly inadequate
bibliography. There are lots of refs in the text, but no
huge list of citations at the end. A proper bibliography
might have changed an 1106-page book into two 750-page
volumes. As thing stand, the publisher probably didn't
make a profit. Gabe's big fat book is seldom cited despite
being full of wisdom. Sic transit gloria.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1

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