Re: [Histonet] Hematoxylin
You did not say which hematoxylin you are using. Harris, Gill, another
progressive formulation i.e. Anatechs hematoxylin??
Blue residue is caused by several things, the worst source is too much
adhesive in a waterbath IF you use a gelatin or egg albumin based section
adhesive. Plus charge slides can also have the problem. Really old
hematoxylin (outdated) can also create some ugly problems including in
If you are using progressive Hematoxylin i.e. Gills formulations, the
background staining on slide is removed very simply with a clarifying rinse
Stain in hematoxylin
running tap water 1 min
30 dips or so in 4% acetic acid (Richard Allen (RA) calls it Clarifier but
it does contain acetic acid and RA recommends 1 minute in their
clarifier). You don't want to leave sections in this solution for a long
time, but some do the rinse for 30 seconds. Whatever works, it is not a
differentiation step but rather a way to reduce background, break up ionic
interactions of dye to slides. ANATECH should have recommendations for
Running tap water 1 min
Bluing (Scotts tap water substitute or RA bluing solution) for 1 minute
Rinse with running tap water 1 minute.
Use fresh clarifier and bluing solutions daily in order to keep things
clean and adjustment of sections to proper pH before eosin
counterstaining. These solutions are cheap compared to having problems
and doing recuts, or just ugly slides. Use adequate rinsing and if you
use standing rinses, change these between slide racks.
If you use Harris hematoxylin, the acid alcohol step should remove
background from slides (unless excessive adhesive is on slides!).
I don't think it is Anatech's hematoxylin that is the problem but rather
other factors needed to fine tune your staining.
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)
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