Anti-CD45RB antibody [EPR31068-523] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45RB antibody [EPR31068-523] - BSA and Azide free (AB326731)

This data was developed using ab326720, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling with ab326720 at 1/2000 (0.256 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in human tonsil.

The primary antibody was incubated 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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45RB antibody [EPR31068-523] - BSA and Azide free (AB326731)

This data was developed using ab326720, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded (A) HEK-293T (human epithelial cell line from embryonic kidney) transfected with a human CD45RB expression vector containing a Myc-His tag. (B) HEK-293T transfected with a human CD45RA expression vector containing a Myc-His tag. (C) HEK-293T transfected with a human CD45RAB expression vector containing a Myc-His tag. (D) HEK-293T transfected with a human CD45RC expression vector containing a Myc-His tag. (E) HEK-293T transfected with a human CD45RO expression vector containing a Myc-His tag. (F) HEK-293T transfected with empty vector containing a Myc-His tag. tissue labeling with ab326720 at 1/10000 (0.051 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in (A) HEK-293T transfected with a human CD45RB expression vector containing a Myc-His tag, and no signal in (B) HEK-293T transfected with a human CD45RA expression vector containing a Myc-His tag, (C) HEK-293T transfected with a human CD45RAB expression vector containing a Myc-His tag, (D) HEK-293T transfected with a human CD45RC expression vector containing a Myc-His tag, (E) HEK-293T transfected with a human CD45RO expression vector containing a Myc-His tag, (F) HEK-293T transfected with empty vector containing a Myc-His tag.

The primary antibody was incubated 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

• Flow Cyt

Lab

Flow Cytometry - Anti-CD45RB antibody [EPR31068-523] - BSA and Azide free (AB326731)

This data was developed using ab326720, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of Human PBMC (human peripheral blood mononuclear cell) cells labelling with ab326720 at 1/500 dilution (0.1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left 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.

Gated on viable lymphoid cells. Cells were co-stained with CD4 conjugated to APC/Fire™ 750.

• Flow Cyt

Lab

Flow Cytometry - Anti-CD45RB antibody [EPR31068-523] - BSA and Azide free (AB326731)

This data was developed using ab326720, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of Human PBMC (human peripheral blood mononuclear cell) cells labelling with ab326720 at 1/500 dilution (0.1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left 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.

Gated on viable cells. Cells were co-stained with CD11c conjugated to Alexa Fluor®647.

• Flow Cyt

Lab

Flow Cytometry - Anti-CD45RB antibody [EPR31068-523] - BSA and Azide free (AB326731)

This data was developed using ab326720, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of Jurkat (human T cell leukemia T lymphocyte from peripheral blood, Right) SH-SY5Y (human neuroblastoma epithelial cell, Left) cells labelling with ab326720 at 1/500 dilution (0.1ug) / Red compared with a Rabbit monoclonal IgG (ab172730) / Black 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.

Gated on viable cells. Negative control : SH-SY5Y.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45RB antibody [EPR31068-523] - BSA and Azide free (AB326731)

This data was developed using ab326720, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human Burkitt's lymphoma tissue labeling with ab326720 at 1/2000 (0.256 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in human Burkitt's lymphoma.

The primary antibody was incubated 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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45RB antibody [EPR31068-523] - BSA and Azide free (AB326731)

This data was developed using ab326720, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human thymoma tissue labeling with ab326720 at 1/2000 (0.256 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in human thymoma.

The primary antibody was incubated 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

Lab

Flow Cytometry - Anti-CD45RB antibody [EPR31068-523] - BSA and Azide free (AB326731)

This data was developed using ab326720, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 0.1% Tween-20 permeabilized 293T cells transfected with a human CD45RB expression vector containing a HA-Myc-tag® (Upper left and middle) 293T cells transfected with an empty expression vector containing a myc-His-tag® (Upper right) 293T cells transfected with a human CD45RAB expression vector containing a HA-Myc-tag® (Lower) 293T cells transfected with a human CD45RO expression vector containing a HA-Myc-tag® (Lower) 293T cells transfected with a human CD45RC expression vector containing a HA-Myc-tag® (Lower) 293T cells transfected with a human CD45RA expression vector containing a HA-Myc-tag® (Lower) cells labelling with ab326720 at 1/500 dilution (0.1ug) compared with a Rabbit monoclonal IgG (ab172730) / Left 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.

Cells were surface stained with isotype control or our antibody. Then fixed with 4% PFA for 10min followed by intracellularly stained with anti-myc tag conjugated to Alexa Fluor® 647.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD45RB antibody [EPR31068-523] - BSA and Azide free (AB326731)

This data was developed using ab326720, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Jurkat (human T cell leukemia T lymphocyte from peripheral blood) SH-SY5Y (human neuroblastoma epithelial cell) cells labelling with ab326720 at 1/2000 (0.256 µg/ml) dilution , followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing membranous and cytoplasmic staining in Jurkat cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Negative control : SH-SY5Y. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab326720 at 1/2000 dilution, followed by ab150120 at 1/1000 dilution.-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 at 1/1000 dilution.

• Flow Cyt

Lab

Flow Cytometry - Anti-CD45RB antibody [EPR31068-523] - BSA and Azide free (AB326731)

This data was developed using ab326720, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of Human PBMC (human peripheral blood mononuclear cell) cells labelling with ab326720 at 1/500 dilution (0.1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left 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.

Gated on viable lymphoid cells. Cells were co-stained with CD3 conjugated to Brilliant Violet 421.

• WB

Lab

Western blot - Anti-CD45RB antibody [EPR31068-523] - BSA and Azide free (AB326731)

This data was developed using ab326720, the same antibody clone in a different buffer formulation.

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

This antibody does not cross-react with overexpressed CD45RA, CD45RC, CD45RO and CD45RAB by western blot.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa); Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) (1 : 5000) (200KDa).

All lanes:

Western blot - Anti-CD45RB antibody [EPR31068-523] (<a href='/en-us/products/primary-antibodies/cd45rb-antibody-epr31068-523-ab326720'>ab326720</a>) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 2:

293T cells transfected with human CD45RB expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 3:

293T cells transfected with human CD45RA expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 4:

293T cells transfected with human CD45RC expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 5:

293T cells transfected with human CD45RO expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 6:

293T cells transfected with human CD45RAB expression vector containing a myc-His-tag® 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: 200 kDa,36 kDa

false

Exposure time: 5s

• WB

Lab

Western blot - Anti-CD45RB antibody [EPR31068-523] - BSA and Azide free (AB326731)

This data was developed using ab326720, the same antibody clone in a different buffer formulation.

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

Low expression : SH-SY5Y, skeletal muscle, cerebellum

The identity of the bands lower than 100 kDa are unknown.

Lanes 1-3 are incubated with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 and lanes 4-6 are incubated with Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes 1-3 : 10 seconds; Lanes 4-6 : 15 seconds

All lanes:

Western blot - Anti-CD45RB antibody [EPR31068-523] (<a href='/en-us/products/primary-antibodies/cd45rb-antibody-epr31068-523-ab326720'>ab326720</a>) at 1/1000 dilution

Lane 1:

KARPAS-299 (human T cell lymphoma cell) whole cell lysate at 20 µg

Lane 2:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate at 20 µg

Lane 3:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Human tonsil tissue lysate at 20 µg

Lane 5:

Human skeletal muscle tissue lysate at 20 µg

Lane 6:

Human cerebellum tissue lysate at 20 µg

Secondary

Lanes 1 - 3:

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

Lanes 4 - 6:

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

Observed band size: 200 kDa,36 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45RB antibody [EPR31068-523] - BSA and Azide free (AB326731)

This data was developed using ab326720, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling with ab326720 at 1/2000 (0.256 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : No staining in human skeletal muscle.

The primary antibody was incubated 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