Tissue/cell: rat brain tissue;4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Blocking buffer (normal goat serum,SLC0005) at 37℃ for 20 min;
Incubation: Anti-phospho-P53(Ser20) Polyclonal Antibody, Unconjugated(SL3703R) 1:200, overnight at 4°C; The secondary antibody was Goat Anti-Rabbit IgG, Cy3 conjugated (SL0295G-Cy3)used at 1:200 dilution for 40 minutes at 37°C. DAPI(5ug/ml,blue,SLC0033) was used to stain the cell nuclei
Blank control (blue line):Hela(fixed with 70% ethanol (Overnight at 4℃) and then permeabilized with 90% ice-cold methanol for 30 min on ice).
Primary Antibody (green line): Rabbit Anti-phospho-P53 (Ser20) antibody(SL3703R),Dilution: 5μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-FITC,Dilution: 1μg /test.