[Histonet] 2 um sections H&E staining

[Gayle Callis]

You 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?

 

 

First is a question.   Why do they require a  2μm thick section in the first
place?    I had a pathologist many years ago fall in love with these very
thin section for all tissues with the same complaint of pale staining.   It
was explained to him that this thickness was excellent for bone marrow and
renal biopsies but too thin for the majority of other tissues.  Simple, you
are slicing through cells much of the time and leaving only cell walls for
staining.  It there isn't enough thickness there, then hematoxylin doesn't
have enough tissue thickness to be "vibrant", and the same for th eosin.
The pathologist went back to the former routine 5μm thick sections.   Some
laboratories do use  4 μm routinely. 

 

Things to try: 

 

1.       If this thickness is required to see basement membranes or marrow
cells

a.      Do not use regressive hematoxylin and eosin, but rather progressive
hematoxylin i.e. Gill III type formulation, and do NOT use any
differentiation solution which can remove hematoxylin. 

b.      Increase the thickness of section by trying 3 μm and 4 μm but use
progressive H&E.  If you don't need 2μm, then go to 4 or 5

c.       Treat sections with 1% periodic acid for 10 min, rinse and then
stain with progressive H&E.  This technique is found in Sheehan and Hrapchak
book.   It might improve the staining with your sections. 

d.