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

[Angela Lamberth via Histonet]

Thank you all very much for your suggestions. I'm going to play around with
a progressive H 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  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@lists.utsouthwestern.edu> wrote:
>
>
> When I cut at 2μm my H 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 For H 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?
> ___
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>


-- 
Angela Lamberth
Histology Technician II
Histology Core Lab
La Jolla Institute for Allergy & Immunology
9420 Athena Circle
La Jolla, CA 92037
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Re: [Histonet] How to get your stains more vibrant when cutting at 2μm?

[Rene J Buesa via Histonet]

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 
 wrote:
 

 When I cut at 2μm my H 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 For H 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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[Histonet] How to get your stains more vibrant when cutting at 2μm?

[Angela Lamberth via Histonet]

When I cut at 2μm my H 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 For H 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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Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet