Fig. 3. Immunohistochemical localization of basement membrane proteins in 12-day-old F9-cell-derived embryoid bodies: cells stably expressing the constructs 1III3-5, 1III3-5mut and 1V1-3 were differentiated into embryoid bodies with 5x10^-8 M retinoic acid, cryosectioned and stained for basement membrane components. As a control, F9 cells carrying the empty expression vector were used. 1III3-5/A and 1III3-5/B represent two different clones from one transfection with the 1III3-5 construct. Bar, 50 µm. Upper panels: staining for perlecan shows disruption of basement membrane formation only in embryoid bodies derived from clones expressing the nidogen binding site (1III3-5/A and /B). Middle panels: double immunofluorescence for laminin-1 and nidogen-1. Cryosections of differentiated 12-day-old embryoid bodies were incubated simultaneously with a rabbit polyclonal antibody against laminin-1 (green) and a rat monoclonal antibody against nidogen-1 (red). Embryoid bodies from clones 1III3-5/A and 1III3-5/B show a separation in the laminin-1 and nidogen-1 staining not seen in those derived from control or 1III3-5 expressing cells. Lower panels: double immunofluorescence for nidogen-1 and nidogen-2. Sections of differentiated 12-day-old embryoid bodies stained with a rabbit polyclonal antibody against nidogen-2 (green) and the rat monoclonal antibody against nidogen-1 (red). Both nidogen isoforms colocalize and nidogen-2 is found in the basement membrane of F9-derived embryoid bodies.