Whoops(ERROR) found ref.re: Methylene Blue-Schiffs (also S. spicer)
From:
Rena Fail <RFail@charleston.net>
<html>
<font size=3>Sorry the first method referenced the art in JHC. I
apologize. With a little digging I found the original article which
is the ref for the simpler method and does NOT include a periodic
acid step.IN the future I will be a little less eager to hit send
until I have checked ref thouroughly.<br>
<br>
Rena Fail<br>
<br>
<br>
Dug deeper in the filesAt 01:33 AM 6/20/01 -0400, you wrote:<br>
<blockquote type=cite class=cite cite>My comments follow the quoted lines
from Rena, which<br>
may be horribly fragmented and mangled by Winders<br>
email software. <br>
<br>
On Tue, 19 Jun 2001, Rena Fail wrote:<br>
> A Schiff's methylene blue will stain DNA purple to red and RNA blue.
Ref: <br>
<br>
> A.J.Garvin,B.J.Hall, R.M.Brissie, and S.S. Spicer, Cytochemical
<br>
> differentiation of nucleic acids with a Schiff's Methylene Blue
<br>
> Sequence. J. of Histochem & Cytochem., Vol. 24 No4, April,
1976, <br>
> pg587 This one uses Bouin's as a mordant , 2% schiff's
for 30 <br>
> min.,running water for 5, 0.5% periodic acid for 5, stain in
1% Schiff's <br>
> for 10 min, wash in running water for 5-10min. stain in methylene
blue pH 6 <br>
> for 1-3 min. dif in 95% deh. clear and mount.<br>
> <br>
> An older method of Dr. Spicer's is in the 3rd edition of the AFIP
manual on <br>
> pg 133<br>
<br>
I haven't checked the JHC paper, but are you sure it includes a <br>
periodic acid step? This would make glycoproteins etc Schiff-<br>
positive in addition to DNA, and why should one want to do that?<br>
<br>
The way the method works is: Bouin fixation for 24 hrs causes
<br>
INCOMPLETE Feulgen hydrolysis of DNA. The resulting aldehydes<br>
are detected with Schiff's reagent, and the RNA is then stained <br>
with methylene blue (or azure A in the "Spicer's Feulgen-azure
A"<br>
method in Lillie & Fullmer, p.175; toluidine blue would also be<br>
OK). <br>
<br>
The INCOMPLETENESS of the nucleic acid hydrolysis is the key<br>
feature of this technique. A complete Feulgen hydrolysis, usually<br>
done these days with 5N HCl (pH about -0.3) at 20C, converts all <br>
the DNA to aldehydes but it also changes all the RNA to small, <br>
soluble fragments, so that no RNA remains to be stained with a <br>
basic dye like methylene blue. Bouin's fluid (pH about +1) for<br>
24 hrs at 20C is a less ferocious acid than either this or the <br>
traditional Feulgen reagent, which was 1.0N HCl (pH zero) at<br>
60C for about an hour. That was the regular method when Spicer <br>
wrote up this now venerable method for DNA + RNA in 2 colours. <br>
<br>
Sam Spicer's histochemical publications span more than 40 years<br>
and include clever and insightful contributions to lectin<br>
histochemistry in the late 1990s. He also gave us the generally<br>
accepted classification and nomenclature of histochemically <br>
discernable mucosubstances, long before the advent of lectins <br>
as useful staining reagents.<br>
<br>
----------------------------------------<br>
John A. Kiernan<br>
Department of Anatomy & Cell Biology<br>
The University of Western Ontario<br>
London, Canada N6A 5C1<br>
kiernan@uwo.ca<br>
<a href="http://publish.uwo.ca/~jkiernan" eudora="autourl">http://publish.uwo.ca/~jkiernan</a><br>
</font></blockquote><br>
</html>