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Sample: Ubiquitin protein at 100ng; Primary: Anti-Ubiquitin (bs-1549R) at 1:300 dilution; Secondary: HRP conjugated Goat-Anti-rabbit IgG(bs-0295G-HRP) at 1: 5000 dilution; Predicted band size: 8.5 kD Observed band size: 8.5 kD

Paraformaldehyde-fixed, paraffin embedded (Mouse pancreas); 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 (Ubiquitin) Polyclonal Antibody, Unconjugated (bs-1549R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (Human skin 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 (Ubiquitin) Polyclonal Antibody, Unconjugated (bs-1549R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat colon tissue); 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 (Ubiquitin) Polyclonal Antibody, Unconjugated (bs-1549R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.

HepG2 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (Ubiquitin) polyclonal Antibody, Unconjugated (bs-1549R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

Blank control (Black line): Molt4 (Black). Primary Antibody (green line):Rabbit Anti-Ubiquitin antibody (bs-1549R) Dilution:1μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody (white blue line): Goat anti-rabbit IgG-AF647 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 room temperature. 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.

Blank control（black line）:HepG2. Primary Antibody (green line): Rabbit Anti-Ubiquitin antibody (bs-1549R) Dilution:1ug/Test; Secondary Antibody : Goat anti-rabbit IgG-AF488 Dilution: 0.5ug/Test. Negative control（white blue line）: PBS Isotype control（orange line）: Normal Rabbit IgG 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.