[Histonet] Hematoxylin

Gayle Callis gcallis <@t> montana.edu
Tue Oct 12 14:46:05 CDT 2004


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hematoxylin (outdated) can also create some ugly problems including in 
tissue section.

If you are using progressive Hematoxylin i.e. Gills formulations, the 
background staining on slide is removed very simply with a clarifying rinse 
after hematoxylin.

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 
their product.

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.
Gayle Callis
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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