Recombinant Anti-PD1 antibody [EPR23119-111] (ab243644)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human tonsil labelling PD1 with ab243644 at 1/500 dilution (1.02 µg/mL) (D), Ki67 with ab16667 at 1/200 dilution (0.15 μg/ml) (B) and PD-L1 with ab228462 at 1/100 dilution (0.52 μg/ml) (C). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Panel A: merged staining of anti-Ki67 (magenta; Opal™690), anti-PD-L1 (red; Opal™570) and anti-PD1 (green; Opal™520) on human tonsil.
Panel B: anti-Ki67 stained on nucleus of proliferating cells.
Panel C: anti-PD-L1 stained on membrane of cells involved in T cell inhibition.
Panel D: anti-PD1 stained on antigen-stimulated T cells.

The section was incubated in three rounds of staining: in the order of ab16667 for 10 mins, ab243644 for 30 mins and ab228462 for 10 mins at room temperature. 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.

Immunohistochemical analysis of paraffin-embedded Human endometrial carcinoma tissue labeling PD1 with ab243644 at 1/500 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human endometrial carcinoma stroma (PMID: 27446374) is observed. 
The section was incubated with ab243644 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

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

This blot was developed using a higher sensitivity ECL substrate. The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 30487606, 31783892).

Exposure time: 48 seconds

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human tonsil labelling PD1 with ab243644 at 1/500 dilution (1.02 µg/mL) (D), CD8 with ab178089 at 1/100 dilution (0.83 μg/ml) (C) and TIM 3 with ab242080 at 1/500 dilution (2.09 μg/ml) (B). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.  

Panel A: merged staining of anti-TIM 3 (green; Opal™690), anti-CD8 (red; Opal™520) and anti-PD1 (gray; Opal™570) on human tonsil.

Panel B: anti-TIM 3 stained on membrane of a subset of immune cells.

Panel C: anti-CD8 stained on membrane of a subset of T cells.

Panel D: anti-PD1 stained on membrane of a subset of lymphocytes.

The section was incubated in three rounds of staining: in the order of ab242080, ab243644 and ab178089 for 30 mins at room temperature. 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human spleen tissue labelling PDI with ab243644 at 1.02 µg/mL (B), PD-L 1 with ab213524 at 1/100 dilution (C) and CD68 with ab213363 at 1/300 dilution (D). Anti-Rabbit and Mouse Polymer HRP was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins. Heat mediated antigen retrieval (Leica ER2, PH9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibodies from the previous round, to avoid any cross-reactivity. 

Panel A: merged staining of anti- PD1 (green, Opal™520), anti- PD-L1 (red, Opal™570) and anti- CD68 (yellow, Opal™690).

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 ab243644, ab213363 and ab213524 for 30 mins at room temperature. 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.

Immunohistochemical analysis of paraffin-embedded Human Hodgkin's lymphoma tissue labeling PD1 with ab243644 at 1/500 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human Hodgkin's lymphoma (PMID: 16819321) is observed. 
The section was incubated with ab243644 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

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling PD1 with ab243644 at 1/500 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Membranous staining on germinal center of human tonsil (PMID: 29042640, PMID: 16819321) is observed.
The section was incubated with ab243644 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

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

This blot was developed using a higher sensitivity ECL substrate. The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 30487606, 31783892). 

Exposure time: 48 seconds