I try to stain frozen muscle section by Sirius red stain, the results were always not consistent. Background of muscle fiber can not be washed clear to be yellow as expected, it was dark yellow, light yellow or even green, various background color appear in different group staining or different type of muscle stain. In addition, most of protocol in reference or online information require to stain muscle in Sirius stain solution for 1 hour, then wash in acetic solution for a while, then alcohol, xylene. I followed those protocol and found it was hard to wash muscle fiber to yellow even if I washed in acetic solution for 1 hour or increased the concentration of acetic acid solution to 0.5%. Finally I stain muscle in Sirius solution just for 2 minutes, then followed the protocol, some time I can get good stain, some time I can't, it were inconsistent. I just wonder what was the problem of my protocol or staining. I attached three images. Please help me.
Any comments would be appreciated.
My protocol is as following:
Sirius Red Stain
Sirius Red F3B (CI 35780) Aldrich 36,554-8 synonym Direct Red 80 0.5gm
Saturated aqueous Picric Acid 500ml
[add a small amount of solid picric acid to ensure saturation: 1.2~1.3% (W/V)]
Expiration date minimum 3 years
Acetic Acid (glacial) 5 ml
Water (DI or tap) 1 liter
Fix frozen sections 30 minutes in Neutral Buffered Formalin(For paraffin sections deparaffinize and bring slides to water)
Wash sections in 3 changes of water
Drain sections well and place into Picro-Sirius solution 2 min
Wash sections in 2 changes of acidified water(5min)
Dehydrate in 3 changes of 100% ethanol (5min)
Clear in xylene and mount in permanent mounting media (5min).
Dr. Xilong Li
Hypertension Division, Internal Medicine
University of Texas Southwestern Medical Center
5323 Harry Hiness Blvd-J4.142
Dallas, TX 75390
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