Anti-MCP1 antibody [RM1100] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCP1 antibody [RM1100] - BSA and Azide free (AB315479)

This data was developed using ab315478, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded : Image A - THP-1 (human monocytic leukemia monocyte) cell pellets treated with 80nM Phorbol-12-myristate-13-acetate (PMA) for 24 hours, then added 100ng/ml Lipopolysaccharides (LPS) for 7 hours, 1µg/ml Brefeldin A was added for additional 3 hours; Image B - Untreated THP-1 cell pellets tissue labeling MCP1 with ab315478 at 1/3000 (0.172 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Cytoplasmic staining on THP-1 cell pellets treated with 80nM Phorbol-12-myristate-13-acetate (PMA) for 24 hours, then added 100ng/ml Lipopolysaccharides (LPS) for 7 hours, 1µg/ml Brefeldin A was added for additional 3 hours (Image A); No staining on untreated THP-1 cell pellets (Image B).
The section was incubated with ab315478 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

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-MCP1 antibody [RM1100] - BSA and Azide free (AB315479)

This data was developed using ab315478, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized THP-1 (human monocytic leukemia monocyte) cells labelling MCP1 with ab315478 at 1/100 (5.17 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing cytoplasmic staining on THP-1 cells treated with 80nM Phorbol-12-myristate-13-acetate (PMA) for 24 hours, then added 100ng/ml Lipopolysaccharides (LPS) for 7 hours, 1µg/ml Brefeldin A was added for additional 3 hours; no staining on untreated THP-1 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 (2.5ug/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 (2 ug/ml) dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCP1 antibody [RM1100] - BSA and Azide free (AB315479)

This data was developed using ab315478, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling MCP1 with ab315478 at 1/3000 (0.172 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on human skeletal muscle.
The section was incubated with ab315478 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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCP1 antibody [RM1100] - BSA and Azide free (AB315479)

This data was developed using ab315478, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded : Image A - Wild-type Hela cell pellets treated with Brefeldin A (1ug/ml) for 3 hours; Image B - Wild-type Hela cell pellets treated with TNF-alpha (TNFα, 20ng/ml) for 3 hours, 1µg/ml Brefeldin A was added for additional 3 hours; Image C - CCL2 knockout HeLa cell pellets treated with Brefeldin A (1ug/ml) for 3 hours; Image D - CCL2 knockout HeLa cell pellets treated with TNF-alpha (TNFα, 20ng/ml) for 3 hours, 1µg/ml Brefeldin A was added for additional 3 hours tissue labeling MCP1 with ab315478 at 1/3000 (0.172 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on Wild-type Hela cell pellets treated with Brefeldin A (1ug/ml) for 3 hours (Image A) and Wild-type Hela cell pellets treated with TNF-alpha (TNFα, 20ng/ml) for 3 hours, 1µg/ml Brefeldin A was added for additional 3 hours (Image B); No staining on CCL2 knockout HeLa cell pellets treated with Brefeldin A (1ug/ml) for 3 hours (Image C) and CCL2 knockout HeLa cell pellets treated with TNF-alpha (TNFα, 20ng/ml) for 3 hours, 1µg/ml Brefeldin A was added for additional 3 hours (Image D).
The section was incubated with ab315478 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

• IP

Supplier Data

Immunoprecipitation - Anti-MCP1 antibody [RM1100] - BSA and Azide free (AB315479)

This data was developed using ab315478, the same antibody clone in a different buffer formulation.

MCP1 was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia monocyte) treated with 80nM Phorbol-12-myristate-13-acetate (PMA) for 24 hours, then added 100ng/ml Lipopolysaccharides (LPS) for 7 hours, 1µg/ml Brefeldin A was added for last 3 hours whole cell lysate with ab315478 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab315478 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

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

All lanes:

Immunoprecipitation - Anti-MCP1 antibody [RM1100] (<a href='/en-us/products/primary-antibodies/mcp1-antibody-rm1100-ab315478'>ab315478</a>) at 1/1000 dilution

Lane 1:

THP-1 (human monocytic leukemia monocyte) treated with 80nM Phorbol-12-myristate-13-acetate (PMA) for 24 hours, then added 100ng/ml Lipopolysaccharides (LPS) for 7 hours, 1µg/ml Brefeldin A was added for last 3 hours whole cell lysate

Lane 2:

<a href='/en-us/products/primary-antibodies/mcp1-antibody-rm1100-ab315478'>ab315478</a> at 1/30 IP in THP-1 (human monocytic leukemia monocyte) treated with 80nM Phorbol-12-myristate-13-acetate (PMA) for 24 hours, then added 100ng/ml Lipopolysaccharides (LPS) for 7 hours, 1µg/ml Brefeldin A was added for last 3 hours whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/mcp1-antibody-rm1100-ab315478'>ab315478</a> in THP-1 treated with 80nM Phorbol-12-myristate-13-acetate (PMA) for 24 hours, then added 100ng/ml Lipopolysaccharides (LPS) for 7 hours, 1µg/ml Brefeldin A was added for last 3 hours whole cell lysate

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

false

Exposure time: 180s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCP1 antibody [RM1100] - BSA and Azide free (AB315479)

This data was developed using ab315478, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded : Image A - RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) cell pellets treated with 100ng/ml Lipopolysaccharides (LPS) for 7 hours, then 1µg/ml Brefeldin A was added for additional 3 hours; Image B - Untreated Raw264.7 cell pellets tissue labeling MCP1 with ab315478 at 1/3000 (0.172 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Cytoplasmic staining on RAW 264.7 cell pellets treated with 100ng/ml Lipopolysaccharides (LPS) for 7 hours, then 1µg/ml Brefeldin A was added for additional 3 hours (Image A); No staining on untreated RAW 264.7 cell pellets (Images B).
The images on the left are applied with Anti-MCP1 antibody [RM1100] (ab315478) at 1/3000 dilution and the images on the right are applied with Anti-MCP1 antibody (ab25124) at 1/1500 dilution.
The section was incubated with ab315478 or ab25124 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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCP1 antibody [RM1100] - BSA and Azide free (AB315479)

This data was developed using ab315478, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse skeletal muscle tissue labeling MCP1 with ab315478 at 1/3000 (0.172 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : ab315478 showed no staining on mouse skeletal muscle (ab), while ab25124 showed non-specific staining on the mouse skeletal muscle.
The image on the left is applied with Anti-MCP1 antibody [RM1100] (ab315478) at 1/3000 dilution and the image on the right is applied with Anti-MCP1 antibody (ab25124) at 1/1500 dilution.
The section was incubated with ab315478 or ab25124 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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCP1 antibody [RM1100] - BSA and Azide free (AB315479)

This data was developed using ab315478, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded : Image A - Mouse lung treated with 1ug/ml Lipopolysaccharides (LPS) and 1ug/ml Brefeldin A for 16 hours; Image B - Untreated mouse lung tissue labeling MCP1 with ab315478 at 1/3000 (0.172 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Cytoplasmic staining on mouse lung treated with 1ug/ml Lipopolysaccharides (LPS) and 1ug/ml Brefeldin A for 16 hours (Image A); nearly no satining on untreated mouse lung (Image B).
The images on the left are applied with Anti-MCP1 antibody [RM1100] (ab315478) at 1/3000 dilution and the images on the right are applied with Anti-MCP1 antibody (ab25124) at 1/1500 dilution.
The section was incubated with ab315478 or ab25124 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

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-MCP1 antibody [RM1100] - BSA and Azide free (AB315479)

This data was developed using ab315478, the same antibody clone in a different buffer formulation.

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 ab315478 at 1/100 (5.17 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing cytoplasmic staining on RAW 264.7 cells treated with 100ng/ml Lipopolysaccharides (LPS) for 7 hours, then 1µg/ml Brefeldin A was added for additional 3 hours; no staining on untreated RAW 264.7 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 (2.5ug/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 (2 ug/ml) dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-MCP1 antibody [RM1100] - BSA and Azide free (AB315479)

This data was developed using ab315478, the same antibody clone in a different buffer formulation.

MCP1 was immunoprecipitated from 0.35 mg RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml Lipopolysaccharides (LPS) for 7 hours, then 1µg/ml Brefeldin A was added for additional 3 hours whole cell lysate with ab315478 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab315478 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

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

All lanes:

Immunoprecipitation - Anti-MCP1 antibody [RM1100] (<a href='/en-us/products/primary-antibodies/mcp1-antibody-rm1100-ab315478'>ab315478</a>) at 1/1000 dilution

Lane 1:

RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml Lipopolysaccharides (LPS) for 7 hours, then 1µg/ml Brefeldin A was added for additional 3 hours whole cell lysate

Lane 2:

<a href='/en-us/products/primary-antibodies/mcp1-antibody-rm1100-ab315478'>ab315478</a> IP in RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml Lipopolysaccharides (LPS) for 7 hours, then 1µg/ml Brefeldin A was added for additional 3 hours whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/mcp1-antibody-rm1100-ab315478'>ab315478</a> in RAW 264.7 treated with 100ng/ml Lipopolysaccharides (LPS) for 7 hours, then 1µg/ml Brefeldin A was added for additional 3 hours whole cell lysate

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

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-MCP1 antibody [RM1100] - BSA and Azide free (AB315479)

This data was developed using ab315478, the same antibody clone in a different buffer formulation.

Western blot : Anti-MCP1 antibody (ab315478) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5](ab8245) loading control staining at 1/20000 dilution, shown in red.

In Western blot, ab315478 was shown to bind specifically to MCP1. Target of interest was observed at 11 kDa in wild-type HeLa cell lysates (lane 1/2) with no signal observed at this size in MCP1 knockout cell line (lane 3/4, knockout cell line ab255372/knockout cell lysate ab263807). To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in a fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged.

All lanes:

Western blot - Anti-MCP1 antibody [RM1100] (<a href='/en-us/products/primary-antibodies/mcp1-antibody-rm1100-ab315478'>ab315478</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervical adenocarcinoma epithelial cell) treated with 1µg/ml Brefeldin A for 3 hours whole cell lysate at 20 µg

Lane 2:

Wild-type HeLa treated with 1µg/ml TNF-alpha (<a href='/en-us/products/proteins-peptides/recombinant-human-tnf-alpha-protein-ab9642'>ab9642</a>) for 6 hours, 1µg/ml Brefeldin A was added for additional 3 hours whole cell lysate at 20 µg

Lane 3:

CCL2 knockout HeLa treated with 1µg/ml Brefeldin A for 3 hours whole cell lysate at 20 µg

Lane 4:

CCL2 knockout HeLa treated with TNF-alpha (<a href='/en-us/products/proteins-peptides/recombinant-human-tnf-alpha-protein-ab9642'>ab9642</a>) for 6 hours, 1µg/ml Brefeldin A was added for additional 3 hours whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (800CW) and Goat Anti-Mouse IgG H&L (680RD) at 1/20000 dilution

Observed band size: 11 kDa

false

• WB

Supplier Data

Western blot - Anti-MCP1 antibody [RM1100] - BSA and Azide free (AB315479)

This data was developed using ab315478, the same antibody clone in a different buffer formulation.

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-MCP1 antibody [RM1100] (<a href='/en-us/products/primary-antibodies/mcp1-antibody-rm1100-ab315478'>ab315478</a>) at 1/1000 dilution

Lane 1:

Untreated THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 2:

THP-1 treated with 80nM Phorbol-12-myristate-13-acetate (PMA) for 24 hours, then added 100ng/ml Lipopolysaccharides (LPS) for 7 hours, 1µg/ml Brefeldin A was 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

Observed band size: 11 kDa,36 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-MCP1 antibody [RM1100] - BSA and Azide free (AB315479)

This data was developed using ab315478, the same antibody clone in a different buffer formulation.

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 28955950).

Panel A is applied with Anti-MCP1 antibody [RM1100] (ab315478) at 1/1000 dilution and panel B is applied with Anti-MCP1 antibody (ab25124) at 1/1000 dilution.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Exposure time : 26 seconds (panel A);180 seconds (panel B).

All lanes:

Western blot - Anti-MCP1 antibody [RM1100] (<a href='/en-us/products/primary-antibodies/mcp1-antibody-rm1100-ab315478'>ab315478</a>) at 1/1000 dilution

Lane 1:

Untreated RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

RAW 264.7 treated with 100ng/ml Lipopolysaccharides (LPS) for 7 hours, then 1µg/ml Brefeldin A was 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 1/100000 dilution

Observed band size: 20 kDa,36 kDa

false