Immunohistochemical analysis of paraffin-embedded human Hodgkin lymphoma tissue labeling Macrophage Inflammatory Protein 1 alpha / CCL3 + CCL3L1 with ab259372 at 1/1000 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human hodgkin Lymphoma (PMID: 31581676). The section was incubated with ab259372 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X100 permeabilized THP-1 (human monocytic leukemia monocyte) cells labeling Macrophage Inflammatory Protein 1 alpha / CCL3 + CCL3L1 with ab259372 at 1/100 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in THP-1 cells treated with 12-myristate 13-acetate (100 nM) for 16 hours then with lipopolysaccharide (100 ng/ml) for 4 hours and with brefeldin A (1μg/ml) for another 3 hours. Tubulin is counterstained using ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (Red). The nuclear counterstain is DAPI (Blue).

Secondary antibody only control: Used PBS instead of priary antibody, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody at 1/1000 dilution.

Flow cytometric analysis of 5% paraformaldehyde-fixed, 90% methanol-permeabilized THP-1 (Human monocytic leukemia monocyte) (treated with 100nM phorbol 12-myristate 13-acetate (PMA) for 16 hours, then 100ng/ml lipopolysaccharide (LPS) for 4 hours, and add 1ug/ml Brefeldin A (BFA) for another 3 hours) (Red)/ Untreated control (Green) cells labeling Macrophage Inflammatory Protein 1 alpha / CCL3 + CCL3L1 with ab259372 at 1/500 dilution (Red / Green) compared with a Isotype control details (ab172730) (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

All lanes : Anti-Macrophage Inflammatory Protein 1 alpha / CCL3 + CCL3L1 antibody [EPR23751-54] (ab259372) at 1/1000 dilution

Lane 1 : Untreated THP-1 (human monocytic leukemia monocyte) whole cell lysate
Lane 2 : THP-1 (treated with 100nM PMA for 16 hours. And then replace PMA with 100 ng/ml LPS (Lipopolysaccharide) for 4 hours, and then 1 µg/ml Brefeldin A was added for the last 3 hours), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

Predicted band size: 10 kDa
Observed band size: 12 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 8 seconds.

Macrophage Inflammatory Protein 1 alpha / CCL3 + CCL3L1 was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia monocyte) (treated with 100nM PMA for 16 hours. And then replace PMA with 100 ng/ml LPS (Lipopolysaccharide) for 7 hours, 1 μg/ml BFA was added for the last 3 hours) whole cell lysate whole cell lysate with ab259372 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259372 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: THP-1 (treated as above) whole cell lysate 3 ug

Lane 2: ab259372 IP in THP-1 (treated as above) whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab259372 in THP-1 (treated as above) whole cell lysate

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

Exposure time: 8 seconds.

All lanes : Anti-Macrophage Inflammatory Protein 1 alpha / CCL3 + CCL3L1 antibody [EPR23751-54] (ab259372) at 1/5000 dilution

Lane 1 : Untreated RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 2 : RAW 264.7 (treated with 100 ng/ml LPS(Lipopolysaccharide) for 4 hours and then 1 µg/ml Brefeldin A was added for the last 3 hours), whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 dilution

Predicted band size: 10 kDa
Observed band size: 12 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 seconds.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X100 permeabilized RAW264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cells labeling Macrophage Inflammatory Protein 1 alpha / CCL3 + CCL3L1 with ab259372 at 1/100 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in RAW 264.7 cells treated with 12-myristate 13-acetate (100 nM) for 16 hours then with lipopolysaccharide (100 ng/ml) for 4 hours and with brefeldin A (1μg/ml) for another 3 hours. Tubulin is counterstained using ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (Red). The nuclear counterstain is DAPI (Blue).

Secondary antibody only control: Used PBS instead of priary antibody, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody at 1/1000 dilution.

Flow cytometric analysis of 5% paraformaldehyde-fixed, 90% methanol-permeabilized RAW264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) ( treated with 100ng/ml lipopolysaccharide (LPS) for 4 hours, and add Brefeldin A (BFA) for another 3 hours(Red)/ Untreated control (Green)) (Red)/ Untreated control (Green) cells labeling Macrophage Inflammatory Protein 1 alpha / CCL3 + CCL3L1 with ab259372 at 1/500 dilution (Red / Green) compared with a Isotype control details (ab172730) (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded THP-1 cells labeling Macrophage Inflammatory Protein 1 alpha / CCL3 + CCL3L1 with ab259372 at 1/1000 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on THP-1 treated with 100nM PMA, LPS and Brefeldin treatment (image A), and negative staining on untreat THP-1 (image B). The section was incubated with ab259372 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

All lanes : Anti-Macrophage Inflammatory Protein 1 alpha / CCL3 + CCL3L1 antibody [EPR23751-54] (ab259372) at 1/1000 dilution

Lane 1 : His-tagged mouse CCL3 recombinant protein (aa24-92) * 2 ,10 ng
Lane 2 : GST/His-tagged human CCL3L1 recombinant protein (aa24-93), 10 ng
Lane 3 : LIF/His-tagged human CCL4 recombinant protein (aa24-92)*2, 10 ng
Lane 4 : His-tagged human CCL18 recombinant protein (aa21-89),10 ng

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

Predicted band size: 10 kDa

Blocking and dilution buffer: 5% NFDM/TBST.

Exposire time: 10 seconds.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling Macrophage Inflammatory Protein 1 alpha / CCL3 + CCL3L1 with ab259372 at 1/1000 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: No staining on human tonsil. The section was incubated with ab259372 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human spleen tissue labeling Macrophage Inflammatory Protein 1 alpha / CCL3 + CCL3L1 with ab259372 at 1/1000 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: No staining on human spleen tissue. The section was incubated with ab259372 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.