Sirius Red stain
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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.
1) De-wax and hydrate paraffin sections.
2) Stain nuclei with Weigert's haematoxylin for 8 minutes, and then wash the slides for 10 minutes in running tap water).
3) Stain in picro-sirius red for one hour (This gives near-equilibrium staining, which does not increase with longer times. Shorter times should not be used, even if the colors look OK.)
4) Wash in two changes of acidified water.
5) Physically remove most of the water from the slides by vigorous shaking.
6) Dehydrate in three changes of 100% ethanol.
7) 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.)