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 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

All lanes : Anti-PD1 antibody [EPR23119-111] (ab243644) at 1/1000 dilution

Lane 1 : Untreated MOLT-4 (human lymphoblastic leukemia T lymphoblast)
Lane 2 : MOLT-4 treated with 500ng/ml Ionomycin and 10ng/ml Phorbol-12-myristate-13-acetate (PMA) for 24 hours

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 32 kDa
Observed band size: 50-55 kDa why is the actual band size different from the predicted?

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

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

All lanes : Anti-PD1 antibody [EPR23119-111] (ab243644) at 1/1000 dilution

Lane 1 : Human tonsil tissue lysate
Lane 2 : Human tonsil tissue lysate treated with PNGase F

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 32 kDa
Observed band size: 50-55 kDa why is the actual band size different from the predicted?

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