Dear Histonetters,

I have had so many requests for this fixative, decided to post it
for those interested.

calcium acetate  0.5g
zinc acetate     5 g
zinc chloride    5 g (store over a dessicant, extremely hygroscopic)

Dissolve salts in 1 liter 0.1M Tris-HCl buffer. Make up buffer, pH 7.4,
and DO NOT readjust pH after salts are added.  Fix up to 24 hours, paraffin
processing.  Fix at room temp, but I do store fixative in refrigerator until
use, and do not consider it a "killer" fixative, in other words, it may
not render infectious diseases "dead" as formalin usually does, prions
excepted, so transferred to 70% ethanol when fixation was complete, and
processed the next night.

Use an alkaline phosphatase kit, incubate antibodies overnight at 4C,
They used Biogenex Optimax wash buffer (excellent!) and Biogenex diluent
appropriate for alkaline phosphatase staining. Levamisole blocking was done,
and I blocked with 4% acetic acid, 15 mins for gut endog alk phos after
rehydration, excellent staining of cells in Peyer's patches attached to
intestine.  YOU DO NOT NEED TO DO ANTIGEN RETRIEVAL!!!!!!!!!!!

H. Nitta et al Cell Vision 1997 4:73-80  Improved in situ
immunodetection of leukocytes on paraffin embedded mouse spleen

Dr. Hiroaki Nitta
Osiris Therapeutics
Baltimore MD

Original publications on this fixative were on human tissue, lymphomas.

J Histochem Cytochem 42:1127-1134, 1994  J.H.Beckstead A simple technique for
preservation of fixation sensitive antigens in paraffin embedded
tissues

J Histochem Cytochem 43:345 JH Beckstead  Same title (addendum)

I strongly recommend you access all these references.  The Nitta et al
gave sources of murine cell surface marker antibodies, and the dilutions
they used.  Very handy, and saved time when working up some of the dilutions.

Happy IHC staining!
Gayle Callis