Re: Picosirius Red stain for collagen

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From:"J. A. Kiernan" <> (by way of histonet)
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On Wed, 17 Nov 1999, Sharon Bledsoe wrote:

> Does anyone have a procedure for picosirius red stain for collagen?

  Here are the relevant few practical paragraphs from my textbook.
  They do not include any discussion that would help you to solve
  unanticipated problems (you'd need to read all of Chapter 8).
  If you are using this stain in research work, you should
  look up the original references. If you will be making measurements
  of phase retardation it will probably be advantageous to use
  half or a quarter of the concentration of sirius red in the
  staining mixture.

Picro-sirius red

The polyazo dye sirius red F3B has much larger molecules than acid
fuchsine, indigocarmine or aniline blue, and it is also able to assume a
planar configuration (see Chapter 5). A solution in picric acid provides
red staining of collagen and a yellow background. In ordinary light the
sections closely resemble those stained by van Gieson's method.
Picro-sirius red is used mainly in conjunction with polarized light
microscopy: the natural birefringence of collagen is greatly enhanced by
the binding of long, aligned molecules of sirius red (Puchtler et al.,
1973; Junqueira et al., 1979). When examined through crossed polars,
collagen fibres stand out brilliantly against a black background. Basement
membranes are only slightly or not at all birefringent because their type
IV collagen molecules are not aligned to form fibres.

Solutions required

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.

(Keeps for at least 3 years and can be used many times.)

B. Acidified water

Add 5 ml acetic acid (glacial) to 1 litre of water (tap or distilled).


1. De-wax and hydrate paraffin sections.
2. (Optional, and not usually done) Stain nuclei with Weigert's
    haematoxylin (see under the van Gieson method, Section 8.2.2),
    then wash the slides for 10 minutes in running tap water.
3. Stain in picro-sirius red (Solution A) for one hour.
4. Wash in two changes of acidified water (Solution B).
5. Dehydrate in three changes of 100% ethanol.
6. Clear in xylene and mount in a resinous medium.


In ordinary bright-field microscopy collagen is red on a yellow
background. (Nuclei, if stained, are black.) The intensity of the red
colour can be measured by microdensitometry to provide estimates of
collagen content in different parts of a tissue (Malkusch et al., 1995;
Kratky et al., 1996). 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 keratohyaline granules and some types of mucus, are stained red
but are not birefringent.

It is necessary to rotate the specimen in order to see all the fibres,
because in any single orientation the birefringence of some 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).


 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1
   FAX (Department): (519) 661-3936

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