[Histonet] How to get your stains more vibrant when cutting at 2μm?

[Angela Lamberth]

Thank you all very much for your suggestions. I'm going to play around with
a progressive H&E when I return from vacation next month. I do have
safranin on hand but will need to order some phloxine to experiment with. I
will probably need to order some additional supplies to make the
hematoxylin. I'm not sure yet if I'll start with Mayer's or Gill's or maybe
even Erlich's.

I should note that cutting at 2 isn't required, but it is desired once they
saw that I can. And since I can, I aim to please! :-)

In addition, I'm looking forward to trying the oven dry method before
coverslipping. A rapid dehydration isn't really possible since I'm working
with a xylene substitute (Pro-Par) and have been battling eosin carryover
but that is a whole different animal for another thread.

Thanks again! I'll report back in a month or 2.

On Wed, Jun 29, 2016 at 10:27 AM, Rene J Buesa <rjbuesa at yahoo.com> wrote:

>
> Angela:
> "Pale" results are the trade-off for great quality very thin "2 µm"
> sections but you can always improve intensity somewhat .
> 1- your "regressive" stain, if it is "modern Harris" has the inherent
> problem of lacking mercury chloride and it is little you can do about.
> Perhaps if you use "progressive Mayer" you will get better results. You
> will not have to differentiate (with the intrinsic "danger" of leaving the
> section too pale) and if used fresh Mayer's can be a good approach.
> 2- as to the counterstain perhaps you should add safranine to the eosin
> (20% safranine + 80% eosin) and will get a darker red.
> 3- try to dehydrate as quickly as possible or even better, wash the
> sections in distilled water and place them in an oven at 60ºC for 10
> minutes and coverslip as usual. You will eliminate any "color wash" due to
> the alcohols.
> If you've not enough "trust" on dry/oven dehydration, try with some
> sections as a test. You will like the method.
> René
>
>
> On Tuesday, June 28, 2016 5:53 PM, Angela Lamberth via Histonet <
> histonet at lists.utsouthwestern.edu> wrote:
>
>
> When I cut at 2μm my H&Es and special stains look pale. How can I get my
> stains to pop or am I stuck with pale looking stains when sectioning that
> thin?
>
> I run manual specials and a manual regressive H&E. For H&E I've tried
> increasing my time in hematoxylin (beyond the manufacturer recommendation),
> diluting my acid alcohol differentiation, and increased time in eosin but
> the slides still lack the vibrancy that many of the postdocs desire.
>
> I use Shandon instant hematoxylin and alcoholic eosin by Thermo. Everything
> else I prepare in house from scratch. Any recommendations?
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-- 
Angela Lamberth
Histology Technician II
Histology Core Lab
La Jolla Institute for Allergy & Immunology
9420 Athena Circle
La Jolla, CA 92037