Anti-PD1 antibody [RM1033]

5 product images

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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), Anti-PD-L1 antibody [SP142] - C-terminal ab228462 anti-PD-L1 used at 1/100 dilution (0.52 μg/ml) and Anti-CD68 antibody [EPR20545] 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 Anti-CD68 antibody [EPR20545] ab213363 and ab309363 for 30 mins, then Anti-PD-L1 antibody [SP142] - C-terminal 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.

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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

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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 (Anti-GAPDH antibody [EPR16891] - Loading Control 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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 60 kDa, 80 kDa

  Exposure time: 125s

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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

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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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed 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. Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/mL dilution.