Re: [Histonet] Eosinophil Staining

From:John Kiernan

Eosinophils stain well with eosin! If used in an alkaline solution (pH 8 to 9) , eosinophils are the only stained objects in most tissues. (Paneth cell granules and tails of spermatozoa also stain but are unlikely to cause confusion.) 

Combining H&E with azure II (a mixture of blue cationic thiazine dyes) does not make much sense because the azure and the haemalum  both colour nuclei blue, albeit for different reasons. You could use haemalum amd eosin or Lillie's azure-eosin (an excellent method, better than H&E, using azure A and eosin B). With these methods eosiniphil granules will be red; cytoplasm and collagen pink, nuclei blue-purple - not the best contrast if you want to see all the eosinophils easily at low magnification.

There is more than one dye called sirius red, and their staining properties are not the same because the coloured anions vary greatly in size. In general, eosinophils stand out in sections stained by any anionic dye that can be used at pH 8-9. Biebrich scarlet is good, because it stays the same colour over a wide pH range. 

John Kiernan
Anatomy, UWO
London, Canada
----- Original Message -----
From: "Griffin-Reyes, Michelle A" 
Date: Friday, October 26, 2007 16:09
Subject: [Histonet] Eosinophil Staining

> I am looking to do a comparative staining study on eosinophils (tissue
> is in FFPE). Among several stains, I plan to include a
> hematoxylin/eosin/azure II stain which will stain eosinophils pink
> (Friend et al. 2000) and a Sirius red stain (Aust et al. 2000).
> Unfortunately, for the H&E/azure II stain, no publications give the
> protocol or specific stain sequence. As for the Sirius red 
> stain, the
> study states that the authors bought the stain from Bayer AG in 
> Germany.I have contacted Bayer and no one seems to know what I 
> am talking about.
> Before I substitute the "Bayer" Sirius red, I want to make sure that
> using another company's stain is appropriate as the protocol is
> described as "Sirius red (500mg) was dissolved in 45 ml aqua 
> bidest, 50
> ml absolute ethanol and 1 ml 1% NaOH; 4ml NaCl at 20% solution 
> was added
> until slight precipitation occurred." My main concern is that 
> the Sirius
> red I can order from other company's state there is little to no
> solubility in ethanol. I am hoping someone will be able to help 
> me in
> regards to these questions. Thank you for your time.
> Michelle Griffin-Reyes
> Comparative Pathology Laboratory
> University of Iowa Hospitals and Clinics
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