The pH adjustment can be as simple as the rinse prior to going into eosin,
make sure you rinse well ( I know we tend to shorten the rinse in tap water
after a bluing solution) in tap water, then rinse the same way (dipping in
70% ethanol or whatever the % alcohol your eosin is dissolved in???)
before going into eosin Y solution.  This should help adjust the pH, and
was a hint given by Jerry Fredenburgh (Richard Allan) in his H&E workshop.

If you are using a commercially prepared mixture, the pH probably has been
adjusted for the eosin, or if you make up your own, acetic acid is added
for that purpose.  Rinsing away the basic bluing solution inadequately may
be part of the problem.

One can also try bluing in warm tap water, rather than a bluing solution,
and have used this frequently with hematoxylin on frozen/IHC methods.  This
would eliminate your basic bluing solution, probably work well.


Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303