[Histonet] Overstaining - Mayers H&E

[Anne van Binsbergen]

Well said John -  *eats grass and leaves speaks to the hand*

AbuDhabiAnnie

2009/12/9 John Kiernan <jkiernan <@t> uwo.ca>

> 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
>  > _______________________________________________
> > Histonet mailing list
> > Histonet <@t> lists.utsouthwestern.edu
> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>



-- 
Anne van Binsbergen (Hope)
Abu Dhabi
UAE