1/50. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Possible cell death inducer, in association with other factors.
Involvement in disease
Genetic variation in PDCD1 is associated with susceptibility to systemic lupus erythematosus type 2 (SLEB2) [MIM:605218]. Systemic lupus erythematosus is a chronic, inflammatory and often febrile multisystemic disorder of connective tissue. It affects principally the skin, joints, kidneys and serosal membranes. It is thought to represent a failure of the regulatory mechanisms of the autoimmune system.
IHC image of PD1 staining in normal human tonsil formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab52587 at 5 µg/ml for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
MOLT4 cells stained for PD-1 (colored green) using ab52587 in ICC/IF. Cells were fixed with 100% methanol (5 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% Tween-20 for 1 hour at room temperature to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the PD-1 antibody ab52587 at 10 µg/ml and ab6046 (Rabbit polyclonal to beta Tubulin - Loading Control) at 1 ug/ml overnight at +4°C. The secondary antibodies used were Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150117) secondary antibody used at 1 ug/ml (colored green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) (ab150080) secondary antibody (pseudo-colored red) used at 2 ug/ml for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1 hour at room temperature.
Sample: Human tonsil cell extract
Dilution: ab52587 antibody was used as 1:200 in 1x106 cells/tube.
Anti-CD4 antibody was used as 1:200 dilution
Anti-CD8 antibody was used as 1:200 dilution
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD1 antibody [NAT105] (ab52587)This image is from PubMedId: 27777963. Kaewkangsadan V et al. (2016)
Immunohistochemical analysis of human large and locally advanced breast cancers staining PD-1 using ab52587. (a) Low level of PD-1+ T cell infiltration; (b) high level of PD-1+ T cell infiltration. (Itu: intratumoral Str: stromal)
Overlay histogram showing MOLT4 cells stained with ab52587 (red line). Live cells were incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab52587, 1/100 dilution) for 30 min at 4°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150117) at 1/2000 dilution for 30 min at 4°C.
A mouse IgG1 isotype control antibody (ab170190) was used at the same concentration and conditions as the primary antibody (black line). Unlabelled sample (blue line) was also used as a control.
Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.
Maruse Y et al. Significant association of increased PD-L1 and PD-1 expression with nodal metastasis and a poor prognosis in oral squamous cell carcinoma. Int J Oral Maxillofac SurgN/A:N/A (2018).
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Balzano T et al. The Cerebellum of Patients with Steatohepatitis Shows Lymphocyte Infiltration, Microglial Activation and Loss of Purkinje and Granular Neurons. Sci Rep8:3004 (2018).
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