[IF=4.718] Arumugam P et al. Expression of a Functional IL-2 Receptor in Vascular Smooth Muscle Cells. The Journal of Immunology,2018 ji1701151.  WB ;  Human.  

PubMed:doi:10.4049/jimmunol.1701151

SHSY5Y 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 (IL-2R gamma) polyclonal Antibody, Unconjugated (bs-2545R) 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.

SHSY5Y 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 (IL-2R gamma) polyclonal Antibody, Unconjugated (bs-2545R) 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: U937(blue).
Primary Antibody: Rabbit Anti-IL-2R,gamma antibody(bs-2545R), 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-2545R, 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.

Black line : Positive blank control (U937); Negative blank control (MCF7）
Green line : Primary Antibody (Rabbit Anti-2R gamma antibody (bs-2545R) )
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 2R gamma Antibody(bs-2545R)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).