Vectashield hard set mounting medium with dapi

Cells were seeded in 400 μl of growth media supplemented with 2% 3D Culture Matrix (Cultrex 3-D Culture Matrix Reduced Growth Factor Basement Membrane Extract, Trevigen, Gaithersburg, MD) in chamber slides. Laminin-V was visualized using anti-laminin-V antibody (#MAB19562, Millipore) followed by secondary antibody (#A21125, Alexa Fluor 594 goat anti-mouse IgG; 1:1000, Molecular Probes) and nuclei were stained with Vectashield Hard Set Mounting Medium with DAPI (Sigma, St. Louis, MO).

After removal of the medium, cells seeded on coverslips were fixed with 4% paraformaldehyde for 15 min for immunocytochemical procedure. The samples were pre-incubated with a blocking solution (10% goat serum and 0.1% Triton X-100 in phosphate-buffered saline) for 30 min and then incubated with a mouse monoclonal anti-GFAP (1:400, Sigma, USA) for 2 hrs at room temperature, followed by incubation with rabbit anti cleaved caspase-3 (Asp175) (1:400, cell signaling) at 4° C overnight. After washes, cells were incubated with the respective secondary antibodies. Finally, after three washings, the sections were mounted with Vectashield HardSet Mounting Medium with DAPI (Sigma). Images were acquired using a Leica confocal microscope.