Anti-MCP1 antibody [EPR27464-89]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCP1 antibody [EPR27464-89] (AB308522)

Immunohistochemical analysis of paraffin-embedded Panel A. Mouse lung tissue labelling MCP1 with ab308522 at 1/500 (1.026 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on mouse lung treated with 1ug/ml LPS for 16h (Panel A); Low expression on untreated mouse lung (Panel B).The section was incubated with ab308522 for 30 mins at room temperature (PMID : 14688348).The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCP1 antibody [EPR27464-89] (AB308522)

Immunohistochemical analysis of paraffin-embedded Mouse skeletal muscl tissue labelling MCP1 with ab308522 at 1/500 (1.026 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : No staining on mouse skeletal muscle. The section was incubated with ab308522 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MCP1 antibody [EPR27464-89] (AB308522)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) cells labelling MCP1 with ab308522 at 1/50 (10.26 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green). Confocal image showing cytoplasmic staining after the treatment with lipopolysaccharide (LPS, 100ng/ml) for 4+3 hours, then Brefeldin A (1ug/ml) for the last 3 h in RAW264.7 cells.Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5 ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

• IP

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Immunoprecipitation - Anti-MCP1 antibody [EPR27464-89] (AB308522)

MCP1 was immunoprecipitated from 0.35 mg RAW264.7 treated with 100 ng/mL LPS for 4 hours, 1 µg/mL Brefeldin A was then added for additional 3 hours whole cell lysate with ab308522 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab308522 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100 ng/mL LPS for 4 hours, 1 µg/mL Brefeldin A was then added for additional 3 hours whole cell lysate Lane 2 : abAB308522 IP in RAW264.7 treated with 100 ng/mL LPS for 4 hours, 1 µg/mL Brefeldin A was then added for additional 3 hours whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab308522 in RAW264.7 treated with 100 ng/mL LPS for 4 hours, 1 µg/mL Brefeldin A was then added for additional 3 hours whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 10 seconds

All lanes:

Immunoprecipitation - Anti-MCP1 antibody [EPR27464-89] (ab308522) at 1/30 dilution

All lanes:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100 ng/mL LPS for 4 hours, 1 µg/mL Brefeldin A was then added for additional 3 hours whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 20 kDa

false

Exposure time: 10s

• IP

Supplier Data

Immunoprecipitation - Anti-MCP1 antibody [EPR27464-89] (AB308522)

MCP1 was immunoprecipitated from 0.35 mg Mouse lung tissue treated with 1 µg/mL LPS and 1 µg/mL Brefeldin A for 16 hours lysate with ab308522 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab308522 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : Mouse lung tissue treated with 1 µg/mL LPS and 1 µg/mL Brefeldin A for 16 hours lysate Lane 2 : abAB308522 IP in Mouse lung tissue treated with 1 µg/mL LPS and 1 µg/mL Brefeldin A for 16 hours lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab308522 in Mouse lung tissue treated with 1 µg/mL LPS and 1 µg/mL Brefeldin A for 16 hours lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 180 seconds

All lanes:

Immunoprecipitation - Anti-MCP1 antibody [EPR27464-89] (ab308522) at 1/30 dilution

All lanes:

Mouse lung tissue treated with 1 µg/mL LPS and 1 µg/mL Brefeldin A for 16 hours lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 20 kDa

false

Exposure time: 180s

• WB

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Western blot - Anti-MCP1 antibody [EPR27464-89] (AB308522)

Blocking/ Dilution buffer : 5% NFDM/TBST. Exposure time : 26 seconds. The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 28955950). The expression of MCP1 is upregulated in response to LPS treatment (PMID : 21444765). In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

All lanes:

Western blot - Anti-MCP1 antibody [EPR27464-89] (ab308522) at 1/1000 dilution

Lane 1:

Untreated RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate, at 20 µg

Lane 2:

RAW264.7 treated with 100 ng/mL LPS for 4 hours, 1 µg/mL Brefeldin A was then added for additional 3 hours whole cell lysate at 20 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 1/20000 dilution

Predicted band size: 11 kDa

Observed band size: 20 kDa

false

Exposure time: 26s