[Histonet] Re #2: μm H&E staining, WHOOPS!!

[Gayle Callis]

Sorry about hitting send too soon. 

 

Repeat of things to try: 

 

a.          Do not use regressive hematoxylin and eosin where hematoxylin
can be overly differentiated i.e. removed from too thin sections.  Use
progressive hematoxylin i.e. Gill II or Gill III type formulation. DO NOT
use acid alcohol differentiation with progressive hematoxylin.  Try staining
longer, i.e. 10 min in Gill III, and use acetic acid clarifier only 1 or 2
dips or skip clarifying solution entirely.     

b.          Never use acid alcohol differentiation even with your
hematoxylin

c.           Use progressive hematoxylin, and do not clarify or use acid
alcohol differentiation solution.  Wash well for 1 minute in running tap
water then blue. 

d.           Increase the thickness of sections to see if this satisfies the
post-docs.  Start with 3 μm and 4 μm but stain these sections with
progressive H&E.  If you don't need 2 μm, then go to a more routine  4 μm or
5 μm thickness.   You need to explain to these post docs about too thin
sections do NOT have enough tissue/cell left to stain well enough.     

e.           Treat sections with FRESH MADE 1% periodic acid for 10 min,
rinse well and stain with progressive H&E.  This periodic acid technique is
found in Sheehan and Hrapchak Theory and Practice of Histotechnology book.
PA treatment might improve the staining with your sections by making more
groups on DNA available to hematoxylin.   However, I didn't find it improved
my thin section staining as much as I wanted.   The sections were just too
thin. 

f.            Try Eosin-phloxine mixture, start dehydration in a few quick
dips in 95%, then proceed to 100% alcohols.   Eosin-phloxine is available as
ready to use or make up in the lab.   Sheehan and Hrapchak is also a source
of this eosin formulation.  

 

Good luck

Gayle M. Callis HT/HTL/MT(ASCP)

GCallis Histology Service, LLC.