Picro-Sirius Red Stain (For Collagen)

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.

Fixation: 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. This protocol has not been tested on frozen sections.
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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.)