Anti-CD147 antibody [EPR18008-8]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD147 antibody [EPR18008-8] (AB188190)

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.

• Flow Cyt

Lab

Flow Cytometry - 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD147 antibody [EPR18008-8] (AB188190)

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.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-CD147 antibody [EPR18008-8] (AB188190)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD147 antibody [EPR18008-8] (AB188190)

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.

• IP

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Immunoprecipitation - Anti-CD147 antibody [EPR18008-8] (AB188190)

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.

All lanes:

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

Predicted band size: 42 kDa

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• sELISA

Lab

Sandwich ELISA - Anti-CD147 antibody [EPR18008-8] (AB188190)

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

• WB

Supplier Data

Western blot - Anti-CD147 antibody [EPR18008-8] (AB188190)

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).

All lanes:

Western blot - Anti-CD147 antibody [EPR18008-8] (ab188190) at 1/5000 dilution

Lane 1:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 2:

Mouse liver lysate at 10 µg

Lane 3:

WEHI-3 (Mouse leukemia cell line) whole cell lysate at 10 µg

Lane 4:

bEnd.3 (Mouse brain endothelioma cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 32 kDa,42 kDa

Observed band size: 45-55 kDa

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• WB

Supplier Data

Western blot - Anti-CD147 antibody [EPR18008-8] (AB188190)

Blocking/Dilution buffer : 5% NFDM/TBST.

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

All lanes:

Western blot - Anti-CD147 antibody [EPR18008-8] (ab188190) at 1/5000 dilution

All lanes:

Mouse CD147 recombinant protein fragment at 0.002 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 42 kDa,72 kDa

Observed band size: 28 kDa

false

Exposure time: 3min

• WB

CiteAb

Western blot - Anti-CD147 antibody [EPR18008-8] (AB188190)

CD147 western blot using anti-CD147 antibody [EPR18008-8] ab188190. Publication image and figure legend from Jin, R., Zhong, W., et al., 2019, J Neuroinflammation, PubMed 31666088.

ab188190 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab188190 please see the product overview.

Focal cerebral ischemia and reperfusion induces CD147 expression and early inflammatory response in the spleen. a Representative western blot images (left) and semi-quantitation (right) showing CD147 protein levels detected in isolated splenocytes from the following groups (n = 5 mice/group) : naïve (no surgery), sham surgery, and tMCAO at 4 or 24 h tMCAO. b The number of isolated splenocytes from each of the above indicated groups was counted using a hemocytometer. c RT-qPCR analysis of CD147 mRNA levels detected in isolated whole splenocytes at 4 or 24 h after tMCAO, and in the different subpopulations of splenocytes (T or B cells neutrophils, monocytes) sorted by FACS at 4 h after tMCAO. *p < 0.05 vs. naïve or sham controls. d RT-qPCR analysis of mRNA levels of proinflammatory genes (TNF-α, IL1β, IL6, MCP-1) detected in isolated splenocytes from the following groups (n = 5 me/group) : sham surgery, tMCAO + isotype, and tMCAO + αCD147. *p < 0.05 vs. sham; #p < 0.05 vs. tMCAO + isotype

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