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

[Rene J Buesa]

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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