CD31 Recombinant Rabbit Monoclonal Antibody [SU03-59]

Western blot analysis of CD31 on THP-1 cell lysates with Rabbit anti-CD31 antibody (ET1608-48) at 1/2,000 dilution.

Lysates/proteins at 15 µg/Lane.

Predicted band size: 83 kDa
Observed band size: 130 kDa

Exposure time: 20 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1608-48) at 1/2,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:50,000 dilution was used for 1 hour at room temperature.

CD31 was immunoprecipitated in 0.2mg THP-1 cell lysate with ET1608-48 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1608-48 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: THP-1 cell lysate (input)
Lane 2: Rabbit IgG instead of ET1608-48 in THP-1 cell lysate
Lane 3: ET1608-48 IP in THP-1 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 5 seconds

Immunohistochemical analysis of paraffin-embedded human tonsil tissue with Rabbit anti-CD31 antibody (ET1608-48) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1608-48) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Flow cytometric analysis of CD31 was done on THP-1 cells. The cells were fixed, permeabilized and stained with the primary antibody (ET1608-48, 1/50) (blue). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; red).

Immunocytochemistry analysis of THP-1 cells labeling CD31 with Rabbit anti-CD31 antibody (ET1608-48) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-CD31 antibody (ET1608-48) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Immunofluorescence analysis of paraffin-embedded human tonsil tissue labeling CD31 with Rabbit anti-CD31 antibody (ET1608-48) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (ET1608-48, green) at 1/200 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).