Alexa fluor 488 conjugated goat anti rabbit igg or goat anti mouse igg

Astrocyte and neuronal cultures were fixed with 4% PFA in phosphate‐buffered saline (PBS; pH 7.4) for 15 min and nonspecific sites were blocked with 3% bovine serum albumin (BSA; Sigma‐Aldrich), 5% normal goat serum (Sigma‐Aldrich) and 0.2% Triton X‐100 diluted in PBS for 1 h, before incubation with the following antibodies: rabbit anti‐GFAP (1:1000; DAKO Cytomation), rabbit anti‐lamin‐B1 (1:1000; Abcam), rabbit anti‐p16INK4a (1:100; Proteintech), rabbit anti‐iNOS (1:100; Abcam), rabbit anti‐Spinophilin (1:500; Abcam), or mouse anti‐Synaptophysin (1:1000; Millipore) at 4°C overnight. Subsequently, the cells were thoroughly washed with PBS and incubated with secondary antibodies at room temperature (RT) for 2 h. Secondary antibodies were Alexa Fluor 546‐conjugated goat anti‐rabbit IgG or goat anti‐mouse IgG (1:1000; Invitrogen), or Alexa Fluor 488‐conjugated goat anti‐rabbit IgG or goat anti‐mouse IgG (1:300; Invitrogen). Nuclei were counterstained with DAPI (Sigma‐Aldrich), and cells were observed with a TE2000 Nikon microscope.