WB
Western blot analysis of PSD95 on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (1/2,000) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody at 1:5,000 dilution was used for 1 hour at room temperature.
Positive control:
Lane 1: SHG-44 cell lysate,
Lane 2: A172 cell lysate,
Lane 3: N2A cell lysate.
ICC/IF
Immunocytochemistry analysis of Neuro-2a cells labeling PSD95 with Mouse anti-PSD95 antibody at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Mouse anti-PSD95 antibody at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (488) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
beta Tubulin (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (594) were used as the secondary antibody at 1/1,000 dilution.
FC
Flow cytometric analysis of PSD95 was done on Hela cells. The cells were fixed, permeabilized and stained with the primary antibody (1/100) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Mouse IgG Secondary antibody at 1/1000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; black).