Anti-RANTES antibody [EPR25836-32] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RANTES antibody [EPR25836-32] - BSA and Azide free (AB324774)

This data was developed using ab322195, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded (A) RAW264.7 treated with 100ng/ml LPS for 4h 1ug/ml BFA was then added for additional 3h cell pellets; (B) Untreated RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cell pellets; tissue labeling RANTES with ab322195 at 1/2000 (0.269 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in A : RAW264.7 treated with 100ng/ml LPS for 4h 1ug/ml BFA was then added for additional 3h cell pellets no staining in B : untreated RAW 264.7 cell pellets.
The section was incubated with ab322195 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-RANTES antibody [EPR25836-32] - BSA and Azide free (AB324774)

This data was developed using ab322195, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling RANTES with ab322195 at 1/100 (5.37 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : No staining in mouse colon.
The section was incubated with ab322195 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-RANTES antibody [EPR25836-32] - BSA and Azide free (AB324774)

This data was developed using ab322195, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse T-cell lymphoma tissue labeling RANTES with ab322195 at 1/100 (5.37 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in mouse T-cell lymphoma.
The section was incubated with ab322195 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 Citrate buffer (pH 6.0 Epitope Retrieval Solution 1) for 20 mins

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-RANTES antibody [EPR25836-32] - BSA and Azide free (AB324774)

This data was developed using ab322195, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml LPS for 4+3 hours then add 1ug/ml BFA for 3 hours (Green); Untreated control (Magenta) cells labelling RANTES with ab322195 at 1/500 dilution (0.1ug) / Magenta and Green compared with a Rabbit monoclonal IgG (ab172730) / Black and Grey isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RANTES antibody [EPR25836-32] - BSA and Azide free (AB324774)

This data was developed using ab322195, 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 RANTES with ab322195 at 1/50 (10.74 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 in Raw 264.7 cells (shown in green) treated with lipopolysaccharides (LPS 100 ng/ml) for 4 hours then add Brefeldin A (1 ug/ml) for another 3 hours . The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). 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 (Magenta). 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RANTES antibody [EPR25836-32] - BSA and Azide free (AB324774)

This data was developed using ab322195, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling RANTES with ab322195 at 1/100 (5.37 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in mouse spleen.
The section was incubated with ab322195 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 Citrate buffer (pH 6.0 Epitope Retrieval Solution 1) for 20 mins

• WB

Supplier Data

Western blot - Anti-RANTES antibody [EPR25836-32] - BSA and Azide free (AB324774)

This data was developed using ab322195, the same antibody clone in a different buffer formulation.

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

Negative control : Raji.

The expression of RANTES is upregulated in response to PMA LPS and BFA treatment.

This blot was developed using a high-sensitivity ECL substrate allowing for the detection of proteins in the mid-femtogram range.

In Western blot Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

All lanes:

Western blot - Anti-RANTES antibody [EPR25836-32] (<a href='/products/primary-antibodies/rantes-antibody-epr25836-32-ab322195'>ab322195</a>) at 1/1000 dilution

Lane 1:

Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 2:

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

Lane 3:

THP-1 treated first with 100ng/ml PMA for 48h, then replaced with 100ng/ml LPS for 21h, 600ng/ml BFA was then added for additional 3h, whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 10 kDa,124 kDa

true

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-RANTES antibody [EPR25836-32] - BSA and Azide free (AB324774)

This data was developed using ab322195, the same antibody clone in a different buffer formulation.

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

The expression of RANTES is upregulated in response to PMA LPS and BFA treatment.

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

All lanes:

Western blot - Anti-RANTES antibody [EPR25836-32] (<a href='/products/primary-antibodies/rantes-antibody-epr25836-32-ab322195'>ab322195</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 LPS for 16h whole cell lysate at 20 µg

Lanes 3 and 5:

Untreated RAW 264.7 whole cell lysate at 20 µg

Lane 4:

RAW264.7 treated first with 80nM PMA for 24h, then replaced with 100ng/ml LPS for 4h, 1ug/ml BFA was then added for additional 3h, whole cell lysate at 20 µg

Lane 6:

RAW264.7 treated with 80nM PMA for 24h whole cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 10 kDa,36 kDa

false

Exposure time: 37s