Mystery:Disappearing melanin!

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From:Greg Dobbin <dobbin@Upei.CA> (by way of histonet)
To:histonet <>
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Hello All (again):

One of our graduate students is working on lobster hemolymph (blood).
She is trying to use cytochemistry to demonstrate phenyloxidase
activity in lobster hemocytes.

The cells are collected into a seawater-based anticoagulant
containing a small amount of formalin.

They are then incubated in a working solution (rx. mixture) of
L-dopa (substrate) and trypsin (activates intracellular enzyme).
Melanin is the expected end product.

She is obtaining nice brown-black staining of intracellular
granules, BUT, the staining disappears when the mounting media is
placed on the cells prior to coverslipping.

The staining survives hematoxylin counterstaining and subsequent tap
water wash, survives the alcohol dehydration, and xylene clearing,
but disappears when mounting media added. She tried both solvent
based and aqueous mounting medias. ANY IDEAS???

                                                       Thanks. Greg
Greg Dobbin
Pathology Lab
Atlantic Veterinary College, U.P.E.I.
550 Unviversity Ave.
Charlottetown, P.E.I.
C1A 4P3
Phone: (902)566-0744
Fax: (902)566-0851

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