Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence

EM of ultrathin cryosections of cells was performed as previously described (Peters et al., 1991a) . Briefly, cells were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 h at 37~ washed in PBS containing 0.15 M glycine, and then embedded in 10% gelatin. Small gelatin blocks were incubated overnight in 2.3 M sucrose at 40C and then frozen in liquid nitrogen. 80-nm thin cryosections were made with a cryo-ultramicrotome (Ultracut S; Reichert Jung, Vienna) using a Diatome diamond knife (Slot et al., 1988) and incubated at room temperature with a polyclonal antibody to Tac (R3134) for 30-60 rain followed by incubation with protein A-gold complexes for 20 min at room temperature. Purified rabbit anti-mouse immunoglobulin (at 2 #g/ml) was used after the rnAb and before protein A-gold. In the case of double labeling with anti-TGN38 antibodies, after the first incubation with gold-conjngate~l protein A, sections were incubated with 1% glutaraldehyde for 10 min and then with PBS containing 0.15 M glycine to prevent interaction between both immunoreagents (Slot et al., 1991) . Before double-labeled sections were evaluated, the specificity of antibody staining was ensured by examining single-labeled sections and by reversing the order of incubations, as previously described (Peters et al., 1991b) . Irrelevant control antibodies did not give any labeling. Sections were examined with a Phillips CM 10 transmission electron microscope.