If the frozen sections are of unfixed tissue, immersion in formaldehyde for 30 min will not fix them. The picric acid in the stain will, by a different mechanism, and that's not what's wanted. I suggest fixing the sections by immersing the slides in 95% alcohol for a minute or two. Take back to water and do the staining method as prescribed=2E This will give you something similar to hydrated paraffin sections. 

The acetic acid in the post-stain rinse is to prevent loss of bound dyes=2C not to differentiate the stain. If you want to remove some bound dye (which should never be necessary with this method), use a neutral liquid such as tap water. With muscle you should see red endomysial collagen surrounding yellow (or unstained) individual muscle fibres.  The yellow colour from the picric acid is variable; it gets extracted into the alcohols used for dehydration after staining. If you prefer to have colourless cytoplasm, simply leave the slides for a long time in the first of the three changes of 100% alcohol.

You didn't say how thick the frozen sections are. If they are thicker than about 20 um they might need a longer wash, with agitation in acidified water. 

John Kiernan
Anatomy, UWO
London, Canada
---
----- Original Message -----
From: Xilong Li 
Date: Thursday, October 25, 2007 11:31
Subject: [Histonet] Sirius Red Stain
To: histonet@lists.utsouthwestern.edu

> Hi, All,
> 
> 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
> 
> 
> Picro-sirius red 
> 
> 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
> 
> Acidified Water
> Acetic Acid 
> (glacial)                         5 ml
> Water (DI or 
> tap)                         1 liter
> 
> Ø Fix frozen sections 30 minutes in Neutral Buffered Formalin(For
> paraffin sections deparaffinize and bring slides to water)
> Ø Wash sections in 3 changes of water
> Ø Drain sections well and place into Picro-Sirius solution 2 min
> Ø Wash sections in 2 changes of acidified water(5min)
> Ø Dehydrate in 3 changes of 100% ethanol (5min)
> Ø 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
> 
> Tel: 214-648-9966(L)
> Fax: 214-648-7902
> 
> 
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> 
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