Sample:
Lane 1: Heart (Mouse) Lysate at 40 ug
Lane 2: Heart (Rat) Lysate at 40 ug
Lane 3: Cerebrum (Mouse) Lysate at 40 ug
Lane 4: Cerebrum (Rat) Lysate at 40 ug
Primary: Anti-Connexin 43 (bs-0651R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 37-45 kD
Observed band size: 45 kD

Sample: Mcf-7 Cell Lysate at 40 ug
Primary: Anti-Connexin(bs-0651R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/10000 dilution
Predicted band size: 42 kD
Observed band size: 43 kD

Sample:
Heart (Mouse) Lysate at 40 ug
Primary: Anti- Connexin 43(bs-0651R)at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 42 kD
Observed band size: 42/48 kD

Sample:
Bladder (Mouse) Lysate at 40 ug
Primary: Anti- Connexin 43(bs-0651R)at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 42 kD
Observed band size: 42/48 kD

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 (Connexin 43) Polyclonal Antibody, Unconjugated (bs-0651R) 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 stomach cancer); 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 (Connexin 43) Polyclonal Antibody, Unconjugated (bs-0651R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructions and DAB staining.

Tissue/cell:U-251 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 (Connexin 43) polyclonal Antibody, Unconjugated (bs-0651R) 1:100, 90 minutes at 37°C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

Tissue/cell: MCF7 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 (Connexin 43) polyclonal Antibody, Unconjugated (bs-0651R) 1:100, 90 minutes at 37°C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

Tissue/cell: rat heart 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,C-0005) at 37℃ for 20 min;
Incubation: Anti-Connexin 43 Polyclonal Antibody, Unconjugated(bs-0651R) 1:200, overnight at 4°C; The secondary antibody was Goat Anti-Rabbit IgG, PE conjugated(bs-0295G-PE)used at 1:200 dilution for 40 minutes at 37°C.

Blank control (blue line):Hela(blue).
Primary Antibody (green line): Rabbit Anti-Connexin 43 antibody(bs-0651R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): F(ab’)2 fragment goat anti-rabbit IgG-FITC.
Dilution: 1μg /test.
Protocol
The cells were fixed with 2% paraformaldehyde (10 min) , then permeabilized with 90% ice-cold methanol for 30 min on ice.Cells stained with Primary Antibody for 30 min at room temperature. The cells were then incubated in 1 X PBS/2%BSA/10% goat serum to block non-specific protein-protein interactions followed by the antibody for 15 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.