Anti-CD45 antibody [EPR28934-536]

• Flow Cyt

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Flow Cytometry - Anti-CD45 antibody [EPR28934-536] (AB317446)

Flow cytometric analysis of J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage) (Right) / HL-1 (mouse atrial muscle cell) (Left) cells labelling CD45 with ab317446 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) (Blue).

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

Low expression : HL-1. Gated on vaible cells.

• Flow Cyt

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Flow Cytometry - Anti-CD45 antibody [EPR28934-536] (AB317446)

Flow cytometric analysis of RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) (Right) / C2C12 (mouse myoblast) (Left) cells labelling CD45 with ab317446 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) (Blue).

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

Low expression : C2C12. Gated on viable cells.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD45 antibody [EPR28934-536] (AB317446)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage) cells labelling CD45 with ab317446 at 1/500 (1.03 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 membrane staining in J774A.1 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Low expression : C2C12
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 2 ug/mL dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [EPR28934-536] (AB317446)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling CD45 with ab317446 at 1/2000 (0.258 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse spleen.
The section was incubated with ab317446 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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [EPR28934-536] (AB317446)

Immunohistochemical analysis of paraffin-embedded Mouse skeletal muscle tissue labeling CD45 with ab317446 at 1/2000 (0.258 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : Low expression on immune cells of mouse skeletal muscle.
The section was incubated with ab317446 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-CD45 antibody [EPR28934-536] (AB317446)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Mouse PBMC (mouse peripheral blood mononuclear cell) cells labelling CD45 with ab317446 at 1/500 (1.03 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 membrane staining in mouse PBMC (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Anti-rat CD3 mouse monoclonal antibody (Alexa Fluor® 647) was used to counterstain tubulin at 1/100 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 2 ug/mL dilution.

• Flow Cyt

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Flow Cytometry - Anti-CD45 antibody [EPR28934-536] (AB317446)

Flow cytometric analysis of Mouse PBMC cells labelling CD45 with ab317446 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) (Blue).

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

Cells were co-stained with anti-CD3 conjugated to Alexa Fluor®647.
Gated on viable cells.

• IP

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Immunoprecipitation - Anti-CD45 antibody [EPR28934-536] (AB317446)

CD45 was immunoprecipitated from 0.35 mg J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage) whole cell lysate with ab317446 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317446 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage) whole cell lysate
Lane 2 : ab317446 IP in J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317446 in J774A.1 whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-CD45 antibody [EPR28934-536] (ab317446) at 1/30 dilution

All lanes:

J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage) 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

• WB

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Western blot - Anti-CD45 antibody [EPR28934-536] (AB317446)

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

Low expression : C2C12, HL-1.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 37917373).

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

All lanes:

Western blot - Anti-CD45 antibody [EPR28934-536] (ab317446) at 1/1000 dilution

Lane 1:

J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage) whole cell lysate at 80 µg

Lane 2:

C2C12 (mouse myoblast) whole cell lysate at 80 µg

Lane 3:

HL-1 (mouse atrial muscle cell) whole cell lysate at 80 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: 200 kDa,124 kDa

true

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-CD45 antibody [EPR28934-536] (AB317446)

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

Low expression : skeletal muscle.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 37917373).

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

All lanes:

Western blot - Anti-CD45 antibody [EPR28934-536] (ab317446) at 1/1000 dilution

Lane 1:

Mouse thymus tissue lysate at 80 µg

Lane 2:

Mouse skeletal muscle tissue lysate at 80 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: 200 kDa,124 kDa

true

Exposure time: 81s