Anti-CD45 antibody [RM1007]

• IHC-P

AbReview80743****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [RM1007] (AB281586)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of paraformaldehyde-fixed 0.3% Triton X-100 permeabilized human tonsil tissue staining with ab281586 at 10 µg/ml concentration. Samples were incubated with the primary antibody for 1 hour at 20°C. Blocking was done using Donkey Serum 10% + 3% BSA for 24 hours at 4°C. Heat mediated antigen retrieval with Citrate pH 6 & TRIS pH 9.

This image is courtesy of an Abreview submitted by Dylan Windell

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [RM1007] (AB281586)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labelling CD45 with ab281586 at 1/4000 (0.138 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human spleen. The section was incubated with ab281586 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 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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CD45 antibody [RM1007] (AB281586)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Human peripheral blood mononuclear cell (PBMC) cells labelling CD45 with ab281586 at 1/500 dilution (0.1ug). Right compared with a Rabbit monoclonal IgG (ab172730)/ Left isotype control. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• ICC

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Immunocytochemistry - Anti-CD45 antibody [RM1007] (AB281586)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Jurkat cells labelling CD45 with ab281586 at 1/50 (11.0 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green) Confocal image showing membranouse staining in Jurkat cells and no staining in 293T cells is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [RM1007] (AB281586)

Immunohistochemical analysis of paraffin-embedded Human colon cancer tissue labelling CD45 with ab281586 at 1/4000 (0.138 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on stromal cells of human colon cancer (PMID : 30713795). The section was incubated with ab281586 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 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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-CD45 antibody [RM1007] (AB281586)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized 293T (Human embryonic kidney epithelial cell, Left) / Jurkat (Human T cell leukemia T lymphocyte, Right) cells labelling CD45 with ab281586 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). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Negative control : 293T. (PMID : 16005866)

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [RM1007] (AB281586)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labelling CD45 with ab281586 at 1/4000 (0.138 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human tonsil. The section was incubated with ab281586 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 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.

• IP

Supplier Data

Immunoprecipitation - Anti-CD45 antibody [RM1007] (AB281586)

CD45 was immunoprecipitated from 0.35 mg Jurkat (human T cell leukemia T lymphocyte), whole cell lysate 10 ug with ab281586 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab281586 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Jurkat (human T cell leukemia T lymphocyte), whole cell lysate 10 ug

Lane 2 : ab281586 IP in Jurkat whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab281586 in Jurkat whole cell lysate

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

Exposure time : 3 minutes

All lanes:

Immunoprecipitation - Anti-CD45 antibody [RM1007] (ab281586)

Predicted band size: 147 kDa

Observed band size: 180 kDa,240 kDa

false

Exposure time: 3min

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [RM1007] (AB281586)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labelling CD45 with ab281586 at 1/2000 (0.275 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse spleen. The section was incubated with ab281586 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 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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-CD45 antibody [RM1007] (AB281586)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Mouse peripheral blood mononuclear cell (PBMC) cells labelling CD45 with ab281586 at 1/50 dilution (1ug). Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [RM1007] (AB281586)

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labelling CD45 with ab281586 at 1/2000 (0.275 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat spleen. The section was incubated with ab281586 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 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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CD45 antibody [RM1007] (AB281586)

Flow cytometry overlay histogram showing mouse splenocytes stained with ab281586 (right), and isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left), used at the same concentration and conditions as the primary antibody.

The cells were incubated in 1x PBS containing 10μg/ml anti CD16/CD32 and 10% normal goat serum to block FC receptors and non-specific protein-protein interactions followed by fixation and permeabilisation with BD Cytofix/Cytoperm™. The primary antibody (ab281586.0) was then added (1x 10⁶ in 100μl at 0.04 μg/ml (1/2,500)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• WB

Lab

Western blot - Anti-CD45 antibody [RM1007] (AB281586)

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

The MW observed is consistent with the literature (PMID : 14715639).

Negative control : 293T (PMID : 22978632).

This blot was developed using a higher sensitivity ECL substrate.

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-CD45 antibody [RM1007] (ab281586) at 1/1000 dilution

Lane 1:

Jurkat (human T cell leukemia T lymphocyte), whole cell lysate at 20 µg

Lane 2:

293T ( human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

Mouse thymus tissue lysate at 20 µg

Lane 4:

Rat thymus tissue 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

Predicted band size: 147 kDa

Observed band size: 180 kDa,240 kDa

false