Sample:
Lane 1: Huvec (Human) Cell Lysate at 30 ug
Lane 2: A431 (Human) Cell Lysate at 30 ug
Primary:
Anti-ZO-1 (bs-1329R) at 1/1000 dilution
Anti-beta-Actin (bs-0061R) at 1/2000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 220 kD
Observed band size: 220 kD

Independently Validated Antibody, image provided by Science Direct, badge number 029577:Formalin-fixed and paraffin embedded human testis labeled with Anti-ZO-1 Polyclonal Antibody, Unconjugated (bs-1329R) at 1:250 followed by conjugation to the secondary antibody.

Black line : Positive blank control (293T); Negative blank control (HL60）
Green line : Primary Antibody (Rabbit Anti-ZO-1 antibody (bs-11320R) )
Orange line：Isotype Control Antibody (Rabbit IgG) .
Blue line : Secondary Antibody (Goat anti-rabbit IgG-AF647)
293T（Positive）and HL60（Negative control）cells (black) were fixed with 4% PFA for 10min at room temperature, permeabilized with PBST for 20 min at room temperature, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with ZO-1 Antibody(bs-1329R)at 1:50 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2% BSA in PBS, followed by secondary antibody(blue) incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).

Blank control: 293T Cells(blue).
Primary Antibody: Rabbit Anti-ZO-1/AF647 Conjugated antibody (bs-1329R-AF647), Dilution: 1μg in 100 μL 1X PBS containing 0.5% BSA;
Isotype Control Antibody: Rabbit IgG/AF647(orange) ,used under the same conditions.
Protocol
The cells were washed twice with phosphate-buffered saline (PBS). The cells were incubated in 1 X PBS containing 0.5% BSA + 1 0% goat serum (15 min) to block non-specific protein-protein interactions followed by the antibody (bs-1329R-AF647, 1μg /1x10^6 cells) for 30 min on ice. Acquisition of 20,000 events was performed.