Dam cy3

After aldehyde quenching with 0.05M NH₄Cl and permeabilization with 0.1% Triton X-100, cells were preincubated with 1% bovine serum albumin (BSA) for 30 min. Successive incubation was performed for 48 h at 4°C in a mixture of anti-MAP2 and anti-GFAP primary antibodies and b-LEA in PBS/BSA 0.1%. This procedure was followed by incubation with a solution of PBS/BSA 0.1% containing the secondary antibodies DAM-Cy3 (polyclonal, anti-mouse IgG, made in donkey; 1:200 dilution; Cy3 fluorochrome; Jackson Immunoresearch Laboratories) and DAR-Cy5 (polyclonal, donkey anti-rabbit IgG conjugated to the indocarbocyanine Cy5, 1:200, Jackson Immunoresearch Laboratories) to reveal monoclonal and polyclonal antibodies, and Alexa-488-labeled streptavidin (1:200, Molecular Probes) for b-LEA lectin cytochemistry. After rinsing, samples were mounted on coverslips with PBS/glycerol and inspected with a TCS-NT (Leica Laserteknik GmbH) laser scanning confocal microscope, to visualize triple fluorescent labeling. The original emission color of fluorochrome conjugated to secondary antibody DAR-Cy5 has been set to blue to facilitate visual inspection of confocal images. All the thickness of cell cultures (about 12 μm) was acquired by digital superimposing of at least 10 serial optical sections.