[IF=3.53] Lee, Tao-Chen, et al. "Comparison of Surface Markers between Human and Rabbit Mesenchymal Stem Cells." PLOS ONE 9.11 (2014): e111390.  Rabbit.  

PubMed:25380245

Sample:
Lane 1: Human A549 cell lysates
Lane 2: Human MCF-7 cell lysates
Primary: Anti-Integrin alpha 4 (bs-0641R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 111 kDa
Observed band size: 150 kDa

Sample:
Lane 1: Normal human Jurkat cell lysates
Lane 2: Jurkat treated with PMA 125ng/ml 30min
Primary: Anti-Integrin alpha 4 (bs-0641R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 111 kDa
Observed band size: 150 kDa

Blank control: U937(blue).
Primary Antibody: Rabbit Anti-Integrin alpha 4 antibody(bs-0641R), Dilution: 1μg in 100 μL 1X PBS containing 0.5% BSA;
Isotype Control Antibody: Rabbit IgG(orange) ,used under the same conditions );
Secondary Antibody: Goat anti-rabbit IgG-PE(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA.
Protocol
The cells were fixed with 2% paraformaldehyde (10 min). Primary antibody (bs-0641R, 1μg /1x10^6 cells) were incubated for 30 min on the ice, followed by 1 X PBS containing 0.5% BSA + 10% goat serum (15 min) to block non-specific protein-protein interactions. Then the Goat Anti-rabbit IgG/PE antibody was added into the blocking buffer mentioned above to react with the primary antibody at 1/200 dilution for 30 min on ice. Acquisition of 20,000 events was performed.

Blank control:THP-1.
Primary Antibody (green line): Rabbit Anti-Integrin alpha 4 antibody (bs-0641R)
Dilution: 2μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-FITC
Dilution: 1μg /test.
Protocol
The cells were 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.

Black line : Positive blank control (U937); Negative blank control (MCF7）
Green line : Primary Antibody (Rabbit Anti-Integrin alpha 4 antibody (bs-0641R) )
Orange line：Isotype Control Antibody (Rabbit IgG) .
Blue line : Secondary Antibody (Goat anti-rabbit IgG-AF647)
U937（Positive）and MCF7（Negative control）cells (black) were incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with Integrin alpha 4 Antibody(bs-0641R)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).