Fig. 1.

Detection of cytoplasmic, secreted, and nuclear clusterin in spinal cord–DRG cultures maintained 3–6 weeks in vitro. a Antibody SC-6419 (produced in goat) detected cytoplasmic clusterin in motor neurons and astrocytes by indirect immunocytochemistry, as well as clusterin secreted into serum-free culture medium by Western analysis. Secondary antibody was cy3-conjugated anti-goat IgG. Motor neurons were identified by their characteristic morphology and astrocytes by double label with rabbit anti-GFAP visualized by cy2-conjugated anti-rabbit IgG. Medium was collected at 24, 48, and 72 h after exchange, clarified by centrifugation, and concentrated using 10-kDa-cut-off Nanosep tubes prior to SDS-PAGE/Western analysis. Albumin, a constituent of the culture medium, was used as loading control. b Antibody SC-8354 (produced in rabbit) detected only nuclear clusterin. Secondary antibody was cy3-anti-rabbit IgG. Specificity of both antibodies was validated by standard preabsorption with recombinant protein (not shown)