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. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet