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Sample: Lane 1: Lymph node (Mouse) Lysate at 40 ug Lane 2: Spleen (Mouse) Lysate at 40 ug Lane 3: Small intestine (Mouse) Lysate at 40 ug Lane 4: NIH/3T3 (Mouse) Cell Lysate at 30 ug Lane 5: Lymph node (Rat) Lysate at 40 ug Lane 6: Spleen (Rat) Lysate at 40 ug Lane 7: Small intestine (Rat) Lysate at 40 ug Lane 8: Bone (Rat) Lysate at 40 ug Lane 9: HL60 (Human) Cell Lysate at 30 ug Lane 10: A431 (Human) Cell Lysate at 30 ug Lane 11: K562 (Human) Cell Lysate at 30 ug Lane 12: MDA-MB-231 (Human) Cell Lysate at 30 ug Lane 13: Raji (Human) Cell Lysate at 30 ug Lane 14: A549 (Human) Cell Lysate at 30 ug Primary: Anti-MCL1 (bs-23315R) at 1/1000 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 39 kD Observed band size: 40 kD

Paraformaldehyde-fixed, paraffin embedded (human lung carcinoma); 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 (MCL1) Polyclonal Antibody, Unconjugated (bs-23315R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (Mouse embryo); 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 (MCL1) Polyclonal Antibody, Unconjugated (bs-23315R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructions and DAB staining.

Paraformaldehyde-fixed, paraffin embedded (human colon carcinoma); 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 (MCL1) Polyclonal Antibody, Unconjugated (bs-23315R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse brain); 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 (MCL1) Polyclonal Antibody, Unconjugated (bs-23315R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Blank control: K562. Primary Antibody (green line): Rabbit Anti-MCL1 antibody (bs-23315R) Dilution: 1μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody : Goat anti-rabbit IgG-FITC Dilution: 1μg /test. Protocol The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at-20℃. The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.