Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
I have been asked to do a "picrosirius" staining procedure. What is it?
Picro-sirius red is a solution of sirius red F3B (0.1%) in saturated aqueous picric acid. It is typically used after an iron haematoxylin nuclear stain, much as Van Gieson, but for 60 minutes. Rinse in slightly acidified water and dehydrate in three changes of absolute alcohol. The result is similar to Van Gieson (Collagen red, cytoplasms & red cells yellow) but sirius red shows thinner fibres that are often missed by Van Gieson. The real difference is seen by using a polarizing microscope. With crossed polars the collagen fibres, even very thin ones, appear in brilliant orange, yellow and green colours against a black background. Basement membranes, though stained, do not exhibit this birefringence because their collagen fibres are not aligned.
The dye isn't one of those certified by the Biological Stain Commission, and some major American vendors do not have it in their catalogues. The stuff in my lab was bought from BDH (Gurr) about 15 years ago. There are many synonyms. The Colour Index application name is Direct red 80, and the CI number is 35780. Don't use a dye that is not CI 35780 even if it has the words sirius and red in its name.
Puchtler H & Sweat F 1964. Histochemie 4, 29-54
Puchtler H, Sweat FS & Valentine LS 1973.
Beitr. Pathol. 150, 174-187
Junqueira LCU, Bignolas G & Brentain RR 1979.
Histochem. J. 11, 447-455
Lillie RD 1977. Conn's Biological Stains, 9th ed.
Baltimore: Williams & Wilkins.
Colour Index CD-ROM (1997) Society of Dyers & Colourists,
John A. Kiernan,