VALIDATION IMAGES
Sample:
Lane 1: Liver (Mouse) Lysate at 40 ug
Lane 2: Stomach (Mouse) Lysate at 40 ug
Lane 3: Kidney (Mouse) Lysate at 40 ug
Lane 4: Spleen (Mouse) Lysate at 40 ug
Lane 5: Muscle (Mouse) Lysate at 40 ug
Lane 6: Small intestine (Rat) Lysate at 40 ug
Lane 7: Hela (Human) Cell Lysate at 30 ug
Lane 8: A549 (Human) Cell Lysate at 30 ug
Lane 9: Panc-1 (Human) Cell Lysate at 30 ug
Primary: Anti-SQSTM1/p62 (bs-55207R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 62 kD
Observed band size: 60 kD
Paraformaldehyde-fixed, paraffin embedded (Mouse liver); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (SQSTM1) Polyclonal Antibody, Unconjugated (bs-55207R) at 1:100 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Rat ovary); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (SQSTM1) Polyclonal Antibody, Unconjugated (bs-55207R) at 1:100 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Human appendix); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (SQSTM1) Polyclonal Antibody, Unconjugated (bs-55207R) at 1:100 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
4% Paraformaldehyde-fixed Hela (H) cell; Triton X-100 at r.t. for 20 min; Antibody incubation with (P62/SQSTM1) polyclonal Antibody, unconjugated (bs-55207R) 1:100, 90 min at 37°C; followed by conjugated Goat Anti-Rabbit IgG antibody (green, bs-60295G-FITC) at 37°C for 90 min, DAPI (blue, C02-04002) was used to stain the cell nuclei. PBS instead of the primary antibody was used as the blank control.
The Hela (H) cells were fixed with 4% PFA (10 min at r.t.) and then permeabilized with 90% ice-cold methanol for 20 min at -20℃,the cells then were incubated in 5%BSA to block non-specific protein-protein interactions (30 min at r.t.).Primary Antibody (green):Rabbit Anti-P62/SQSTM1 antibody (bs-55207R,1:100); Secondary Antibody (white blue): Goat anti-Rabbit IgG-BF488 (bs-60295G-BF488): 1 μg/test. Blank control (black): PBS. Acquisition of 20,000 events was performed.