[Histonet] thionin staining

John Kiernan jkiernan <@t> uwo.ca
Fri Feb 4 11:42:07 CST 2011

If this is for Nissl staining, the pH of the thionine solution should be 3.5 to 4 and the following rinses should be no more acidic than that. Distilled water is suitable. 
Alcohol-water mixtures generally remove dyes to a greater extent than 100% alcohol.  I shake the washed slides to get rid of most of the water, then go straight to the first of 3 changes of 100% ethanol. Almost no blue is lost from the sections. The first alcohol isn't 100% any more after one or two batches of slides have gone through, so for large numbers of slides it is more economical to go through 95% (quickly) then 3X100%. If you use 50% and 70% alcohol you can expect dye to be extracted from the stained sections.
It's not unusual to get unsatisfactory batches of thionine, and for at least one purpose, showing the canaliculi and lacunae of bone, the late Russ Allison found that proper staining could be obtained only with batches of thionine that had been certified by the Biological Stain Commission.  See Allison RT (1995) Picro-thionin (Schmorl) staining of bone and other hard tissues. Brit. J. Biomed. Sci. 52: 162-164.  The B.S.C.'s tests for thionine are a mast cell stain and a stain for plant tissue infected with a fungus. The dye must also meet spectrophotometric criteria. See Penney DP (2002) Analysis and testing of biological stains - the Biological Stain commission Procedures. Biotech. Histochem. 77:237-275. A minor revision to the criteria for thionine was published in 2008: Lyon HO & Kiernan JA (2008) Notes from the Biological Stain Commission. Biotech. Histochem. 83(5):285-288. 
Make sure your dye really is thionine (CI 52000). There is a dye called thionine blue (CI 52025, Basic blue 25) that is not a substitute. See Conn's Biological Stains or Bryan Llewellyn's StainsFile http://stainsfile.info/StainsFile/dyes/52000.htm .  Finally, the word thionine is commonly mis-spelled, without its terminal e. The e should be there because thionine is an amine, in contrast to eosin, which is not. Dictionaries (English or US) give the correct spelling.
John Kiernan
Anatomy, UWO
London, Canada
= = =
----- Original Message -----
From: An Eerdekens <An.Eerdekens <@t> med.kuleuven.be>
Date: Friday, February 4, 2011 3:21
Subject: [Histonet] thionin staining
To: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu>

> Dear all,
> I have problems with thionin staining. I am using this on 
> paraffin slices of hypothalami. In the past, I never experienced 
> problems, but the last times, the staining is washed off by 
> going through the ethanol after staining (50%, 70%, 90%, 100%). 
> I tried already a few protocols, but every time the same result.
> Does anybody have experience with it?
> regards
> An Eerdekens
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