Localization of platinum in biological cells. enriched in photoreceptor synapses. From this fraction, a Triton X-100-resistant subfraction was purified that consisted primarily of synaptic ribbons and their disassembly products. The high enrichment of synaptic ribbons was verified by electron microscopy and immunolabeling using an antibody that specifically binds to synaptic ribbons. SDS-PAGE analysis of this synaptic ribbon portion displayed several major polypeptide bands migrating at 240, 60,?55,?43,?and 30?kDa. The purification process described here is a 1st promising step toward the recognition of the yet unfamiliar constituents of synaptic ribbons from photoreceptor synapses and possibly also of presynaptic densities from additional synapses. Bovine eyes were obtained from a local slaughterhouse. MC1568 Eyes were transported to the laboratory on ice, and the retinae were isolated and processed within 30?min postmortem. Retinae from adult Wistar rats were prepared within 10?min postmortem. A human being retina was from the Division of Ophthalmology (University or college of Wrzburg) approximately MC1568 60?min after an attention enucleation surgery of a patient who also had an ocular melanoma. A mouse monoclonal antibody directed against synaptophysin, a kind gift from Dr. B.?Wiedenmann (Wiedenmann and Franke, 1985), was used at a 1:100 dilution in PBS, pH 7.4.?A mouse monoclonal antibody against glial fibrillary acidic protein (GFAP) (clone G-A-5) was purchased from Sigma (Deisenhofen, Germany) and used at a dilution in PBS of 1 1:100 for immunofluorescence and 1:10,000 for immunoblotting. A monoclonal antibody against SP14 (Honer et al., 1993; Simpson et al., 1994), which is probably a SNAP25-like molecule (for a review, observe Rothman, 1994), was from Biermann (Bad Nauheim, Germany) and utilized for immunoblotting at a dilution of 1 1:200 in PBS. The antibody used MC1568 in this study for labeling photoreceptor synaptic ribbons (observe below) was present in the serum of a rabbit that had been immunized against the rat erythrocyte anion exchanger (AE1, band 3) (Drenckhahn et al., 1985). The component of synaptic ribbons that was recognized by this antiserum in the retina was not related to AE1, but was attributable to an autoantibody directed against a yet unfamiliar retinal molecule for MC1568 the following reasons. (1) Affinity purification of the serum with AE1 slice out from nitrocellulose bedding after electrophoretic transfer (Olmstedt, 1981) abolished staining of the OPL but not staining of erythrocytes; (2) preabsorption of the serum with these AE1-comprising nitrocellulose sheets eliminated labeling of erythrocytes but not of synapses; and (3) additional polyclonal rabbit antisera directed against AE1 of rat, pig, and human being erythrocytes did not stain photoreceptor synapses, although they brightly stained erythrocytes. For simplicity, this serum that labeled synaptic ribbons is called ribbon antiserum in the following text. Key experiments were carried out with both unfractionated serum and serum preabsorbed with rat AE1 (observe above). The success of preabsorption was checked by immunolabeling of rat blood smears with preabsorbed and nonabsorbed ribbon antiserum. The ribbon antiserum was used at a 1:50 dilution in PBS, pH 7.4. Retinae were processed for immunocytochemistry by flash-freezing in liquid nitrogen-cooled isopentane. Cryostat sections (5?m in thickness) were slice having a Frigocut 2800?E (Reichert-Jung, Nu?loch, Germany). Cryosections were thawed on poly-l-lysine-coated coverslides and dried on a heating plate (37C) for 1?hr. Incubation with main antibodies (diluted as indicated above) was performed over night at 4C. After several washes with PBS to remove unbound antibody, the binding of the primary antibody was visualized with goat anti-rabbit or goat anti-mouse secondary antibodies conjugated to either tetramethyl rhodamine isothiocyanate or fluorescein isothiocyanate (Biotrend, Cologne, Germany) at a Agt 1:100 dilution in PBS, pH 7.4?(1?hr, 22C). 1.5% Aliquots of the different retinal tissue fractions were preserved from each step of the fractionation procedure and fixed by adding glutaraldehyde to a final concentration of 5% (v/v). Fixation was performed for 3?hr at 4C with gentle agitation of the samples. After fixation, samples were sedimented by a 10,000??spin in an Eppendorff centrifuge for 10?min. After several washes with PBS, the pellets were post-fixed with 1% OsO4 in H2O (w/v) for 1?hr at 4C. The samples were block-contrasted with 2% uranyl acetate in H2O (w/v) for 3?hr at 4C, dehydrated with an ascending ethanol concentration series, and embedded inside a resin combination containing 49.6% glycid ether (1,2,3-propanetriol glycidyl ether, epoxy equivalent of 150) (w/w), 21% 2-dodecenylsuccinic acid anhydride (w/w), 29% (w/w) methylnadic anhydride, and 0.4% (w/w) 2,4,6-tris(dimethylamino-methyl)phenol (Serva, Heidelberg, Germany). The resin was polymerized at 60C for 12?hr. Sections were slice having a Reichert Ultracut E (Reichert-Jung). Sections were analyzed having a Zeiss EM109 and photographed with Agfa Ortho 25?film (Agfa-Gevaert, Leverkusen, Germany). A preembedding protocol was utilized for the ultrastructural localization of the antigen recognized from the ribbon antiserum. For immunolabeling, both cryostat sections from whole retina and cryostat sections from retinal fractions prepared as explained above were used. Twenty-micrometer-thick cryostat sections were thawed on poly-l-lysine-coated coverslides. These sections either were processed directly for immunostaining or 1st softly fixed with 0.1% formaldehyde.