Immunocytochemistry analysis of NIH/3T3 cells treated with 0.5μM doxorubicin for 20 hours labeling p53 with Rabbit anti-p53 antibody (HA722074) at 1/200 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 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 Rabbit anti-p53 antibody (HA722074) at 1/200 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) 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 (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

p53 was immunoprecipitated in 0.2mg NIH/3T3 treated with 0.5μM doxorubicincell for 24 hours cell lysate with HA722074 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA722074 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature. Lane 1: NIH/3T3 treated with 0.5μM doxorubicincell for 24 hours cell lysate (input) Lane 2: HA722074 IP in NIH/3T3 treated with 0.5μM doxorubicincell for 24 hours cell lysate Lane 3: Rabbit IgG instead of HA722074 in NIH/3T3 treated with 0.5μM doxorubicincell for 24 hours cell lysate Blocking/Dilution buffer: 5% NFDM/TBST Exposure time: 1 minute; ECL: K1802