Recombinant Anti-CD147 antibody [EPR18008-8] (ab188190)

Flow cytometry staining of C57 BL/6 mouse splenocytes (top) or C57 BL/6 mouse thymocytes (bottom), with ab188190 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left). Splenocytes or thymocytes were incubated for 30 min at 4°C in 1x PBS containing 10μg/ml anti CD16/CD32 and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by ab188190 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 10⁶ in 100 µl at 0.1 μg/ml (1/21900)) for 30 min at 4°C. The cells were simultaneously stained with CD3.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 4°C

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on viable cells.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lane 1/2: 3 minutes; Lane 3: 10 seconds; Lane 4: 1 second.

The expression profile observed is consistent with what has been described in the literature (PMID: 16721788; 23966157).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized bEND.3 (Mouse brain capillary endothelial cell line) cells labeling CD147 with ab188190 at 1/250 dilution, followed by by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cell membrane staining on bEND.3 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab188190 at 1/250 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded mouse intestine tissue labeling CD147 with ab188190 at 1/8000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Membrane staining on mouse intestine is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

CD147 was immunoprecipitated from 1 mg of RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate with ab188190 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab188190 at 1/5000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: RAW 264.7 whole cell lysate 10µg (Input).

Lane 2: ab188190 IP in RAW 264.7 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab188190 in RAW 264.7 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 1 second.

Flow cytometric analysis of fresh mouse thymocytes labeling CD147 with ab188190 at 1/200 dilution (red) compared with Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730; black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat Anti-Rabbit IgG (Alexa Fluor^® 488) at 1/500 dilution was used as the secondary antibody.

Standard Curve for CD147 (Analyte: Recombinant mouse CD147 protein) dilution range 0-500 pg/mL using Capture antibody at 0.2 ug/mL and Detector Antibody at 0.5 ug/mL. Secondary antibody: Peroxidase Streptavidin SA-HRP at 1/20000 dilution. Concentration of ab188190 may vary from lot to lot; please use this curve as guideline.

Washing buffer: 1X PBST
Blocking/Diluting buffer and concentration: 1% BSA/PBS

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized WEHI-231 (Mouse B Cell Lymphoma cell line) cells labeling CD147 with ab188190 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cell membrane staining on WEHI-231 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab188190 at 1/250 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Blocking/Dilution buffer: 5% NFDM/TBST.

Recombinant protein fragment Mouse CD147 contains aa140-325 with a His-Tag^®. It was made in house