Human recombinant PD-L1 (Peprotech), nivolumab (anti-PD-1 monoclonal antibody, Bristol Myers Squibb), pembrolizumab (anti-PD-1 monoclonal antibody, Merck), atezolizumab (anti-PD-L1 monoclonal antibody, Genentech), trastuzumab (anti-HER2 monoclonal antibody, Genentech), daratumumab (anti-CD38 monoclonal antibody, Janssen), and trametinib (anti-MEK1/2 small molecule, Novartis) were obtained. Treatment assays with PD-L1 were performed at a concentration of 1 µg/ml as described.11 (link) The following antibodies were used for western blot assay and immunofluorescence (IF): anti-PD-1 (Proteintech), anti-PD-L1 (Abcam), anti-phospho and total ERK (Cell Signaling), anti-GAPDH (Santa Cruz), and anti-beta-actin (Sigma Aldrich). PD1 (PDCD1) (NM_005018) Human Overexpression Lysate (Origene) and empty vector cell lysate (Origene) were used as PD-1 positive and negative controls, respectively.
Anti pd 1
Manufactured by Proteintech
Sourced in China
Anti-PD-1 is a laboratory reagent used in research applications. It functions as an antibody that binds to the PD-1 protein, which is involved in immune system regulation. This product can be utilized in various in vitro and in vivo studies related to immune system processes and mechanisms.
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Lab products found in correlation
Anti pd l1, by Proteintech (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti phospho and total erk, by Cell Signaling Technology (1 mentions)
Atezolizumab, by Genentech (1 mentions)
Anti pd l1, by Abcam (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Pembrolizumab, by Merck Group (1 mentions)
Trastuzumab, by Genentech (1 mentions)
Trametinib, by Novartis (1 mentions)
Nivolumab, by Bristol-Myers Squibb (1 mentions)
Daratumumab, by Johnson & Johnson (1 mentions)
Lymphocyte separation solution, by Merck Group (1 mentions)
Anti cd8a, by Thermo Fisher Scientific (1 mentions)
Anti cd4, by Thermo Fisher Scientific (1 mentions)
Anti foxp3, by Thermo Fisher Scientific (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Anti ipmk, by Proteintech (1 mentions)
Anti cd206, by Proteintech (1 mentions)
Anti ptx3, by Proteintech (1 mentions)
Immpress anti rabbit ig peroxidase polymer detection kit, by Vector Laboratories (1 mentions)
5 protocols using anti pd 1
1
Immunotherapy Assay Protocols
2
Isolation and characterization of T cell subsets
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Spleens of recipient rats were collected, grounded, and filtered to obtain cell suspension, which was centrifuged at 1600 rpm at 4°C for 5 mins. The precipitation was resuspended by pre-cooled phosphate buffer solution. Double volume of lymphocyte separation solution purchased from Sigma-Aldrich was added and centrifuged at 2000 RPM for 25 min to obtain cell suspension. CD4+ T cells and CD8+ T cells were isolated by LS column (Miltenyi Biotec, Germany, #130-042-401) with the method of antibiotic microbeads (Miltenyi Biotec, Germany, # 130-090-319, # 130-090-318) as previously described (37 (link), 38 (link)). Sorted cells were incubated directly with diluted fluorochrome-conjugated monoclonal antibodies as shown below: anti-CD4 (Invitrogen, #11-0040-82, FITC), anti-CD8a (Invitrogen, #11-0084-82, FITC), anti-PD-1 (Proteintech, #65211, Coralite647) and anti-Foxp3 (eBioscience, #12-5773-82, PE).
3
Immunohistochemical Analysis of Breast Cancer
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After being fixed in the 10% formalin for 24 h, BC samples were sectioned at 4 μm. Immunohistochemistry (IHC) was performed with anti-IPMK (1:200, Proteintech, China), anti-PD-1 (1:100, Proteintech, USA), anti-PD-L1 (1:200, Proteintech, USA) and anti-CTLA-4 (1:100, Proteintech, USA) antibodies as primary antibodies according to the manufacturer’s protocols. For immunofluorescence (IF), anti-IPMK (1:200, Proteintech, USA) and anti-CD206 (1:100, Proteintech, USA) were used as primary antibodies to show the coexpression of CD206 and IPMK in the BC samples.
4
Immunohistochemical Analysis of Glioma
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Patients (n = 60) undergoing the neurosurgical removal of glioma (WHO grade II‐IV) in Xiangya Hospital, Central South University, were the tissue sources (Table S1 ). Slices (4 μm) from glioma (WHO grade II‐IV) tissues were fixed by formalin and embedded in paraffin. Sections were then boiled with sodium citrate buffer for antigen retrieval, and 3% H2O2 was used for blocking endogenous peroxidase activity. A 5% BSA was used for section blocking. Rabbit polyclonal anti‐PTX3 (1:50; Proteintech; Wuhan, China), anti‐HAVCR2 (1:500; Proteintech; Wuhan, China), anti‐PD‐1 (1:400; Proteintech; Wuhan, China), anti‐PD‐L1 (1:5000; Proteintech; Wuhan, China), and anti‐CD276 antibody (1:400; Proteintech; Wuhan, China) were used as the primary antibody. Sections were incubated at 4°C overnight. Horseradish peroxidase (HRP) conjugated secondary antibody and 3,3′‐diaminobenzidine were used for visualization. Hematoxylin was used for counterstaining with an optical microscope used for observation. H‐score was calculated by intensity score * quantity score. As for intensity scores, 0, 1, 2, and 3 represented negative, weak, moderate, and strong, respectively. The quantity score was determined by the proportion of stained cells, in which 0, 1, 2, 3, and 4 represented <10%, 10%–25%, 25%–50%, 50%–75%, and >75%, respectively. The H‐score had a range of 0–12.
5
Quantifying Immune Checkpoint Markers in ESCC
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Immunohistochemical analysis of MARCH7 in ESCC (n=25) and distant matched non-malignant tissues (n=24) was performed as described before [15] . Additionally, immunohistochemical analysis of CD8 (cluster of differentiation 8) and PD-1 (programmed cell death protein-1) expression in ESCC tissues was also carried out. Brie y, following depara nization and rehydration oftissue sections antigen retrieval was carried out using Tris-EDTA buffer (10 mM Tris-base, 1 mM EDTA, pH 9.0). Then, slides were treated with diluted rabbit polyclonal anti-MARCH7 (1:100; Novus Biologicals, USA), anti-CD8 (1:100; Proteintech, USA) and anti-PD-1 (1:100;Proteintech, USA) antibodies of human origin and incubated overnight at 4 C.
Next day, the slides were treated with HRP conjugated anti-rabbit IgG [ImmPRESS Anti-Rabbit Ig (peroxidase) Polymer Detection Kit, Vector Laboratories Inc, USA] for further analysis.
Next day, the slides were treated with HRP conjugated anti-rabbit IgG [ImmPRESS Anti-Rabbit Ig (peroxidase) Polymer Detection Kit, Vector Laboratories Inc, USA] for further analysis.
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