[Histonet] Overstaining - Mayers H&E

Edwards, Richard E. ree3 <@t> leicester.ac.uk
Thu Dec 10 08:50:11 CST 2009

Spot on!.

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of John Kiernan
Sent: 09 December 2009 17:10
To: Josephine Garcia
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Overstaining - Mayers H&E

This is a typical example of the informal "protocols" that get passed on from generation to generation of graduate students, postdocs and technicians in research labs at universities. The original was probably written by someone who knew how to do H&E staining, but on differently fixed tissues, and certainly on thinner sections. It apears to be for individual slides, because 10 seconds in each of the two 95% and 100% ethanols would be effective only with vigorous agitation in a large excess of fluid.
The tissue is almost unfixed, unless "Soak in 0.4% paraformaldehyde"  means leave it ovenight or longer in 4% formaldehyde. 
Researchers otherwise educated to the highest levels in such difficult disciplines as molecular biology and neuroscience regularly write phrases like "4% paraformaldehyde", thereby advertising their profound ignorance about fixation, which is the procedure that has the greatest effect on the appearance of anything dead that's examined with a microscope, especially if stains or histochemical methods are to be used. (I apologise for the length of the preceding sentence, but not for its punctuation, which is correct in British but not in American English usage. Check it out with Lynne Truss!)
The "sucrose cycle" step, with no times or instructions about floating and sinking, is probably local jargon from a lab where small animals' brains are minimally fixed and cryoprotected before cutting thick (50-100um) frozen sections, to be stained free-floating. That's not an H&E job! You are working with a thin skeletal muscle (rat's gastrocnemius).
If your Mayer's haemalum is a bought solution, it is intended for use in hospital labs, on paraffin sections about 5um thick. In a research setting you may need to make changes. Haemalum (Mayer's or anyone else's, correctly used) should stain cell nuclei blue and very little else. 
An important part of H&E staining is looking at the wet section with a microscope to check for adequate and selective nuclear coloration. In skeletal muscle the nuclei are small, so the haemalum-stained section is very pale blue to the unaided eye. With alcoholic eosin (as in your method) it's not so easy to control the staining with microscopic control, but it will probably be OK if the section is light pink. Some people like their eosin darker; it's largely a matter of taste unless you need to distinguish between different eosinophilic components on the basis of hue. 
John Kiernan
Anatomy, UWO
London, Canada
= = =
-----Original Message -----
From: Josephine Garcia <jbdg <@t> u.washington.edu>
Date: Monday, December 7, 2009 11:43
Subject: [Histonet] Overstaining - Mayers H&E
To: histonet <@t> lists.utsouthwestern.edu

> Hi all,
> My (frozen-section, fixed) slides are coming out much too dark 
> (overstainedpurple) and I'm not sure why. They are 15-20 
> micrometer slices of rat
> gastrocnemius muscle. Can someone please look over our current 
> protocol and
> tell me what I'm doing wrong? Thanks! Here it is:
> 1. Perfuse animal with 4% paraformaldehyde fixative.
> 2. Soak in 0.4% paraformaldehyde
> 3. Sucrose cycle (5% rinse, 10%, 20%, 30% soak)
> 4. Embed in OCT, Frozen sections (15-20 micrometers)
> 5. Let dry for 15-30 min
> 6. Stain as follows:
> - Distilled H2O (quick dip)
> - Mayer's Hematoxylin - 1min (originally we were dipping for 5-
> 10 minutes. I
> slowly reduced the time to 2min, then 1min, then 30s... still 
> overstained!)- Running lukewarm tap water - drain and 
> continuously fill - 15min or until
> water runs clear
> - Distilled H2O (quick dip)
> - 80% EtOH - 1-2min
> - Eosin - 2 min
> - 95% EtOH I - 10sec
> - 95% EtOH  II - 10sec
> - 100% EtOH I - 10sec
> - 100% EtOH II - 10sec
> - Xylene I - 2min
> - Xylene II - 2min
> 7. Coverslip and let dry overnight
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