Anti-PD1 antibody [RM1033]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD1 antibody [RM1033] (AB309363)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling PD1 with ab309363 at 1/4000 (0.125 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : no staining on human cerebrum. The section was incubated with ab309363 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 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-PD1 antibody [RM1033] (AB309363)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling PD1 with ab309363 at 1/4000 (0.125 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human tonsil. The section was incubated with ab309363 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• mIHC

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Multiplex immunohistochemistry - Anti-PD1 antibody [RM1033] (AB309363)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human tonsil tissue staining PD1 with ab309363 at a 1/4000 dilution ( 0.125 μg/ml), ab228462 anti-PD-L1 used at 1/100 dilution (0.52 μg/ml) and ab213363 anti-CD68 used at a 1/500 dilution (1.26 μg/ml).

Panel A : merged staining of anti-CD68 (gray; Opal™690), anti-PD1 (green; Opal™520) and anti-PD-L1 (red; Opal™570) on human tonsil.
Panel B : anti-PD1 stained on antigen-stimulated T cells.
Panel C : anti-PD-L1 stained on cells involved in T cell inhibition.
Panel D : anti-CD68 stained on macrophages.

The section was incubated in three rounds of staining : in the order of ab213363 and ab309363 for 30 mins, then ab228462 for 10 mins at room temperature.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PD1 antibody [RM1033] (AB309363)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized EL-4 (mouse lymphoma T lymphocyte) cells labelling PD1 with ab309363 at 1/50 (10.02 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 membranous and cytoplasmic staining in EL-4 cell line.Negative control : CTLL-2 (PMID : 1396582).Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). 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.

• WB

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Western blot - Anti-PD1 antibody [RM1033] (AB309363)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Negative control : CTLL-2 (PMID : 1396582). In Western blot, anti-GAPDH antibody (ab181602) staining at 1/20, 0000 dilution. Exposure time : 125 seconds

All lanes:

Western blot - Anti-PD1 antibody [RM1033] (ab309363) at 1/1000 dilution

Lane 1:

MOLT-4 (human lymphoblastic leukemia T lymphoblast) whole cell lysate at 20 µg

Lane 2:

EL4 (mouse lymphoma T lymphocyte) whole cell lysate at 20 µg

Lane 3:

EL4.IL-2 (mouse lymphoma T lymphocyte) whole cell lysate at 20 µg

Lane 4:

CTLL-2 (mouse T lymphocyte) whole cell lysate at 20 µg

Lane 5:

Human tomsil 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

Observed band size: 60 kDa,80 kDa

false

Exposure time: 125s