[Histonet] RE: Re: Methanol in H2O2 explanation

Wed Jul 11 12:18:16 CDT 2012

Now that's funny! Even if it makes we PhDs look bad. 

I was once given some tissue in "formaldehyde" from one of my colleagues in another department to process and stain. The resulting slides looked horrendous and I suspected the fix was the problem. I went back to their lab and asked to see the bottle of "formaldehyde" which turned out to be formamide. Then I got the shoulder shrug and "We thought it was close enough" response. Arrrrrgghh.

Brett

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Geoff McAuliffe
Sent: Wednesday, July 11, 2012 12:50 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] RE: Re: Methanol in H2O2 explanation

I agree, lots of "lore" gets passed down without question or understanding.

Many years ago a new PhD who had trained in a good lab came to my lab 
looking for some "NaH." I tried to explain that there was no such 
chemical when she produced a xeroxed lab protocol calling for NaH to 
make a malete buffer for EM (that shows you how long ago this was!). So 
I found the exact recipe but with NaOH. The techs all knew it was a typo 
but she did not.

Geoff

On 7/11/2012 12:12 PM, Morken, Timothy wrote:
> It's funny how histology lore gets passed down, passed around and misunderstood over the decades. When I ask questions in labs about why they do a certain procedure they can rarely name a source (besides a particular long-retired particular tech who wrote the original procedure) or give reason as to why it is done that way. Even if it works well, at least people could take the time to understand why!
>
> When I was researching buffer compositions long ago I found a dozen variations of PBS alone (and only a dozen because I just stopped looking). When you delve into these things it becomes clear that people made their own solutions for a particular purpose. Maybe they wrote a paper, taught a course or wrote a book. Then that formula was copied by many others and their particular formulation became the "state of the art" even if the "art" practiced by someone else had little to do with the original pursuit (the Sheehan book is full of variations of stains that were originally for specific research purposes. There is precious little guidance in any books about what is good for a particular use!).
>
> The reason for using methanol / H2O2  that I was told long ago was prevent frozen sections from being damaged by H2O2 bubbling. In fact, we would fix frozens in cold acetone and THEN put the slides in cold methanol/H2O2 (double fixation?)  In that lab we never used methanol for deparaffinized sections. Since I had no exposure to the clin lab I never knew about using methanol for blood smear fixation. From literature searches it seems methanol, rather than ethanol, is used primarily because it is cheaper. In other words, it does not appear to be a magical substance!
>
> The questions asked here are good ones. However, hopefully those reading and responsible for producing procedure manuals will include the reasoning for each step of the method and not just assume it is necessary, or "common knowledge," just because it has been handed down through generations of long suffering techs!
>
>
> Tim Morken
>
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gudrun Lang
> Sent: Wednesday, July 11, 2012 7:17 AM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: AW: [Histonet] RE: Re: Methanol in H2O2 explanation
>
> For the enzymatic activity of peroxidase it needs an electron-donator (or receptor - I can't find the literature...) in the vicinity; therefore H2O2 and DAB are added ad once, and DAB is oxidized and transformed into the insoluble, amorphe substance through polymerization.
> Without the donator H2O2 in excess works as inhibitor and blocks the activation-side of the enzyme.
> I think H2O2 in methanol was primarly preferred, because the frozen slides were fixed at the same time.
>
> Rehydration after dewaxing depends on the following reagens.
>
> Gudrun
>
> -----Ursprüngliche Nachricht-----
> Von: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von Tony Henwood (SCHN)
> Gesendet: Mittwoch, 11. Juli 2012 06:21
> An: 'Hobbs, Carl'; histonet <@t> lists.utsouthwestern.edu
> Betreff: [Histonet] RE: Re: Methanol in H2O2 explanation
>
> Hi Carl,
>
> What do you mean by "Why do people rehydrate after dewaxing?" Do you really mean that slides do not require rehydration or do you mean that slides can be left to dry after de-waxing prior to staining.
>
> Re-hydration is necessary, otherwise xylene will prevent aqueous stains from doing their thing efficiently.
>
> I was lead to believe that as H2O2 was catalysed by endogenous peroxidase, the reactive oxygen reacted with the methanol to then degrade the enzyme, but I need to look closer at this chemistry.
>
>
> Regards
> Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager&  Senior Scientist
> Tel: 612 9845 3306
> Fax: 612 9845 3318
> the children's hospital at westmead
> Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA
>
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Hobbs, Carl
> Sent: Wednesday, 11 July 2012 1:32 PM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Re: Methanol in H2O2 explanation
>
> Why do some people use methanolic H2O2?
> Why do people rehydrate after dewaxing?
> Both are unnecessary, under usual conditions.
>
> Methanol was used in the early days as a peroxidase blocker by itself.
> The combination was devised as a "belt and bracer" method.
> As you stated, you use aq H2O2 effectively.
> So do I and many others.
>
> However, for unfixed frozen sections, I would never use aq H2O2, if I wanted sections remaining on my slides....
>
> After dewaxing, rinse sections in 4x 100% alcohol, rinse in water, endog. Px block while you make up you AR solutions....
>
> Carl Hobbs
> Histology and Imaging Manager
> Wolfson CARD
> School of Biomedical Sciences
> Kings College London
> Guys Campus
> SE1 1UL
> Tel: 020 78486813
> Fax: 020 78486816
> 020 78486813
>
>
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-- 
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Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
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