Sirius red collagen stain (Long reply)

From:"J. A. Kiernan" <jkiernan@julian.uwo.ca>

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).

Procedure

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
solution.

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.

Result

  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
  black background.

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
  listed below. 

  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
  too long.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1





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