[Histonet] Sirius Red Stain

[John Kiernan]

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. 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, 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 <Xilong.Li <@t> UTSouthwestern.edu>
Date: Thursday, October 25, 2007 11:31
Subject: [Histonet] Sirius Red Stain
To: histonet <@t> 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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