Abstract
Melanin synthesis in melanocytes is localized to melanosomes, where enzymatic and structural gene products must be targeted. Some gene products are translocated through different pathways (i.e., tyrosinase travels through the Golgi apparatus during processing, whereas a putative matrix glycoprotein does not). To visualize trafficking routes, we used post-embedding immunocytochemistry to compliment DOPA-histochemistry. We report on the localization of tyrosinase, matrix glycoprotein, tyrosinase-related protein 1 (TRP-1) and lysosome-associated membrane protein-1 (LAMP-1).DOPA histochemistry: Cultured melanoma cells were fixed in half-strength Karnovsky’s, incubated in 0.1 % 1-DOPA 2X for 2.5 h and embedded in Eponate 12. For immunocytochemistry, cells were fixed with 4% paraformaldehyde, 1% glutaraldehyde, and 0.2% picric acid with 0.5 mM CaCl2(pH 7.4) for 20 m at RT, quenched in 50 mM NH4Cl for 20 m at 0°C, washed in 0.1 M maleate buffer (pH 6.5), and treated with 2% uranyl acetate (pH 4.0) for 30 m at 0°C.
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