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>
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