Sirius red collagen stain (Long reply)
|From:||"J. A. Kiernan" <firstname.lastname@example.org>|
On Mon, 30 Oct 2000, Andrea Grantham wrote:
> I'm trying to find a procedure for a collagen stain using Sirius Red. Has
> anybody done this stain?
Yes! It's one of the best understood techniques of collagen
histochemistry. Technical details follow, and are followed
by some comments and a few references. You should come to
grips with the theory, advantages and limitations of this
method before using it on a large scale.
Picro-sirius red method (after Puchtler et al., 1973;
Junqueira et al., 1979). Step 4 is an addition that
prevents the loss of dye that happens if the
stained sections are washed in water.
Solution A. Picro-sirius red
Sirius red F3B (C.I. 35782): 0.5 g
Saturated aqueous solution
of picric acid: 500 ml
Add a little solid picric acid to ensure saturation
(This is important).
(Keeps for at least 3 years and can be used many times.)
Solution B. Acidified water
Add 5 ml acetic acid (glacial) to 1 litre of
water (tap or distilled).
Fixation is not critical, The method is most frequently used on
paraffin sections of objects fixed adequately (at least 24 hours
but ideally 1 or 2 weeks) in a neutral buffered formaldehyde
1. De-wax and hydrate paraffin sections.
2. (Optional, and not usually done) Stain nuclei with
Weigert's haematoxylin (as for the van Gieson method,
but more strongly, then wash the slides for 10 minutes
in running tap water).
3. Stain in picro-sirius red (Solution A) for one hour.
(This gives near-equilibrium staining, which does not
increase with longer times. Shorter times should not
be used, even if the colours look OK.)
4. Wash in two changes of acidified water (Solution B).
5. Physically remove most of the water from the slides
by vigorous shaking or (for a few slides only)
blotting with damp filter paper.
5. Dehydrate in three changes of 100% ethanol.
6. Clear in xylene and mount in a resinous medium.
In bright-field microscopy collagen is red on a pale yellow
background. (Nuclei, if stained, are ideally black but may
often be grey or brown. The long time in picro-sirius red
causes appreciable de-staining of the nuclei. This is
not a problem with traditional van Gieson or with
picro-aniline blue, with their 1-minute staining times.)
When examined through crossed polars the larger collagen
fibers are bright yellow or orange, and the thinner ones,
including reticular fibers, are green. According to
Junqueira et al. (1979) the birefringence is highly
specific for collagen. A few materials, including Type 4
collagen in basement membranes, keratohyaline granules
and some types of mucus, are stained red but are not
birefringent. It is necessary to rotate the slide in
order to see all the fibres, because in any single
orientation the birefringence of some fibres will be
extinguished. This minor inconvenience can be circumvented
by equipping the microscope for use with circularly rather
than plane polarized light (Whittaker et al., 1994;
Whittaker, 1995), but then you don't get a completely
Comments and References
Although this method is technically very easy, it is important
for the person doing it and (if it's someone else) the person
using the stained slides, to know what it does and how it works.
Even without a polarizing microscope, picro-sirius red shows
things like reticular fibres and the basal laminae of cerebral
capillaries, which are missed by van Gieson and may be obscured
by masses of other stained details in trichrome methods (Mallory,
Masson, Heidenhain etc). To the best of my knowledge, most users
of picro-sirius red are doing research that exploits the
enhancement by sirius red of the birefringence of collagen
fibres, which is largely due to co-aligned molecules of Type I
collagen. It is also used to stain amyloid.
If you are using only polarized light it does not matter if you
lose the "yellow background" of picric acid staining. If you
use picro-sirius red as a "better" van Gieson and want to keep
the yellow cytoplasm, be hasty with the dehydrating - even more
so than with the original van Gieson method.
About 4 years ago, someone (sorry, I've forgotten who, so I
can't shout your name) posted to HistoNet an excellent
bibliography of staining methods using sirius red F3B. This
should be findable in the Archives (www.histosearch.com)
Nobody should do (or order to be done) a picro-sirius red
stain without reading at least one of the first two items
Junqueira LCU, Bignolas G, Brentani RR. Picrosirius staining
plus polarization microscopy, a specific method for collagen
detection in tissue sections. Histochem J 1979; 11, 447-455
Puchtler H, Waldrop FS, Valentine LS. Polarization microscopic
studies of connective tissue stained with picro-sirius red FBA.
Beitr Path 1973; 150, 174-187
Whittaker P. Polarized light microscopy in biomedical research.
Microscopy and Analysis 1995; 44, 15-17
Whittaker P, Kloner RA, Boughner DR, Pickering JG. Quantitative
assessment of myocardial collagen with picrosirius red staining
and circularly polarized light. Basic Research in Cardiology
1994; 89, 397-410
Finally, it's important to get the right dye. Sirius red F3B
is C.I. 35782 (Direct red 80). __There are other "sirius red"s
that are quite different.__ At least one that I've used a lot
is OK but does not carry any C.I. designation on the label. With
this kind of dye (a tetra-azo direct cotton dye) the manufacturing
process necessarily generates more than one coloured product,
and other compounds are added to precipitate the dye and adjust its
colour intensity. Test your sirius red on sections of muscle, brain
and kidney before using it for research or diagnosis. In normal
kidney the glomerular basement membranes should be red but not
birefringent. Every muscle fibre should be surrounded by red and
birefringent collagen. I could continue, but this is already
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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