Tumors were zinc-fixed, paraffin-embedded, and subsequently stained in a fluorescent multiplex immunohistochemistry staining using the OpalTM 7-Color Fluorescent Immunohistochemistry (IHC) Kits (Akoya, Marlborough, USA). The following anti-mouse antibodies were used: anti-αSMA (Sigma-Aldrich, Schnelldorf, Germany F3777), anti-DIO2 (Elabscience, Houston, USA, E-A-13198), anti-GSN (Biozol, Eching, Germany, BOB-PA2109), anti-MMP3 (Santa Cruz, Heidelberg, Germany, sc-21732), anti-Pan-Cytokeratin (Abcam, ab27988), anti-PD-L1 (Cell signaling, D5V38), and anti-PDK4 (Antibodies-online, Aachen, Germany, ABIN3028963) in an automated staining using the BOND RX Automated IHC Research Stainer (Leica Biosystems, Nussloch, Germany). Stained tumor sections were scanned using Vectra® 3 automated quantitative pathology imaging system and analyzed using inForm® software V2.3 (both Akoya). Marker expression in the cytoplasm was quantified with the inForm® software using a positivity or 4-bin (0–3+) scoring algorithm (22 (link)). For the latter spectrally unmixed fluorescence signals in the cytoplasm of epithelial or stromal cells were grouped into four bins based on signal distribution (0 = lowest signal, 3 = highest signal), indicating differences in protein expression.
Anti pd l1
Manufactured by Cell Signaling Technology
Sourced in United States, China
Anti-PD-L1 is a laboratory reagent used for the detection and study of the PD-L1 protein. PD-L1 is a cell surface protein that plays a role in regulating the immune response. Anti-PD-L1 can be used in various immunological techniques, such as Western blotting, immunohistochemistry, and flow cytometry, to identify and characterize PD-L1 expression in different cell types and samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (5 mentions)
Anti stat1, by Cell Signaling Technology (4 mentions)
Anti cd8, by Abcam (4 mentions)
Anti phospho stat1, by Cell Signaling Technology (3 mentions)
Anti hsp90, by Cell Signaling Technology (3 mentions)
Anti akt, by Cell Signaling Technology (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
Anti phospho akt, by Cell Signaling Technology (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Anti gapdh, by Abcam (3 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Anti pd 1, by Cell Signaling Technology (3 mentions)
Opal automation mif detection kit, by Akoya Biosciences (3 mentions)
Anti panck, by Abcam (3 mentions)
Anti cd68, by Abcam (3 mentions)
Anti cd163, by Abcam (3 mentions)
Anti muc1 c, by Thermo Fisher Scientific (3 mentions)
E1l3n, by Cell Signaling Technology (3 mentions)
Anti pd 1, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
76 protocols using anti pd l1
1
Multiplex Immunohistochemistry for Tumor Analysis
2
Immunohistochemistry and Immunofluorescence Protocols
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The protocols used for IHC and immunofluorescence were performed according to previous studies [22 (link)]. Anti-GBE1 (1:300; Abcam, USA), anti-PD-L1 (1:300; Cell Signaling Technology, USA), CD8 (1:300; Abcam, USA), CCL5 (1:300; BioLegend, USA), CXCL10 (1:300; Ruiyingbio, China) were used as primary antibodies. For IHC, three fields of view per sample were imaged. The intensity of immunostaining was taken into consideration when analyzing the data. The percentage scoring of immunoreactive tumor cells was as follows: 0 (< 10%), 1 (10–40%), 2 (40–70%), and 3 (> 70%). The staining intensity was visually scored and stratified as follows: 0 (negative), 1 (yellowish), 2 (light brown), 3 (dark brown). The intensity of staining was obtained by multiplying the two items into a total score, and the scores ranged from 0 to 9. In immunofluorescence, Cy3- and FITC-conjugated secondary antibodies (BioLegend, California, USA, 1:500) were used to detect the primary antibodies. Nuclear staining was performed with DAPI (1:1000; Roche, USA). The samples were visualized with a fluorescence microscope (Olympus, IX71, Japan).
3
Protein Expression and Immunoblotting
4
Immunohistochemical Analysis of Activated Cytotoxic T Cells
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Triple immunohistochemistry staining was used to assess the distribution of activated cytotoxic CD8 T cells. Briefly, brain sections were prepared for immunostaining via xylene treatment and gradual rehydration with 99–75% ethanol. Sections were blocked with 1% FBS for immunohistofluorescence staining or 3% hydrogen peroxide for immunohistochemistry and then incubated with anti-CD8 (#ab17147; Abcam, USA), anti-CD38 (#ab108403; Abcam, USA), anti-HLA-DR (#17221-1-AP; Proteintech, USA), anti-PD-L1 (#13684; Cell Signaling, USA), or anti-CCL5 (#SC-1410; Santa Cruz, USA) overnight at 4°C in blocking solution. The sections were then incubated with fluorescent secondary antibodies or peroxidase-conjugated secondary antibodies for 1 h at room temperature and subsequently stained using 3,3' diaminobenzidine and counterstained with hematoxylin. Slides were coverslipped using mounting medium (Histokitt, Germany). Images were captured with a 40x objective on a Nikon Eclipse E400 microscope using SPOT software (Nikon, Japan).
5
Immunoblot Analysis of Protein Targets
6
Comprehensive Protein Expression Analysis
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Total protein was extracted from tissue or cultured cells using RIPA (Beyotime, China) containing a protease inhibitor cocktail (Roche, USA). Protein concentrations were assessed using a BCA Protein Assay Kit (Beyotime, China). Equal amounts of protein were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, USA). After blocking the membranes with 5% nonfat milk, different specific antibodies were incubated with the membrane. The antibodies used were as follows: anti-PD-L1 (Cell Signaling Technology, 3127S), anti-PI3K (Santa Cruz Biotechnologies cat.no. sc-136298), anti-p-PI3K (Cell Signaling Technology, cat. no. 4228), anti-AKT (Cell Signaling Technology, 4691S), anti-p-AKT (Cell Signaling Technology, 4060S),anti-Bcl-2(Cell Signaling Technology, 15071T), anti-Bax(Cell Signaling Technology, 5023T), and anti-GAPDH (Proteintech, 60004-1-Ig). Goat anti-Mouse HRP (FDbio science, FDM007), and Goat anti-Rabbit HRP (FDbio science, FDR007). Finally, an ECL chromogenic substrate was applied for detecting the fluorescent signals (BIO-RAD, USA).
7
Epithelial-Mesenchymal Transition Markers
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The following antibodies were used in this study: anti-E-cadherin, anti-N-cadherin, anti-vimentin, and anti-PD-L1 from Cell Signaling Technology (Danvers, MA, USA); and anti-β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant human transforming growth factor (TGF)-β1 was purchased from PeproTech (Rocky Hill, NJ, USA). SB 431542, a TGF-β inhibitor, was purchased from Tocris Bioscience (St. Louis, MO, USA).
8
Immunohistochemical Analysis of Immune Checkpoints in Prostate Cancer
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In line with our previous study,30 (link) briefly, formalin-fixed PCa tissue sections were paraffinized in xylene and hydrated in a diluted alcohol series. Then, antigen retrieval was accomplished with sodium citrate buffer (10 mM, pH 6.0) using a microwave for 10 min. After that, the sections were immersed in H2O2 (0.3%) for 15 min to reduce activity of endogenous peroxidase. Following blocking with goat serum, the sections were incubated with corresponding primary antibodies (anti-PD-L1: Cell Signaling Technology (CST) #13684; anti-B7-H3: CST, #14058; anti-HHLA2: clone 566.129 (link); anti-CD8: CST, #85336; anti-Foxp3: CST, #12653) at 4℃ overnight. Then, after washing for three times using phosphate buffered saline (PBS), the sections were subsequently incubated with secondary antibody for 1 hour. Finally, the sections were visualized using a DAKO EnVision Detection System (Dako) and counterstained with hematoxylin.
9
Protein Expression Profiling by Western Blot
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Protein lysis, separation and transfer were performed as previously described (19 (link)). Blots were probed using antibodies against β-actin (Santa Cruz Biotechnology Inc.) as a housekeeping gene, anti-CD84 (Santa Cruz Biotechnology Inc.), anti–phospho AKT (Cell Signaling Technology), anti–phospho S6 (Cell Signaling Technology), and anti–PD-L1 (Cell Signaling Technology). The bands were then detected using ECL Western Blotting Substrate.
10
PD-L1 Expression Analysis by Western Blot
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Western blotting (WB) was performed as described previously38 (link). HepG2 cells were transfected with pCMV-PD-L1 (Sinobio, Wayne, PA, USA), and SK-Hep1/SK-Hep1R cells were treated with IFN-γ. After incubation for 48 h, proteins were extracted using lysis buffer. Protein concentrations were measured and validated using a Nanodrop spectrophotometer (DeNovix Inc, Wilmington, USA), and 20 µg of protein was used for WB. Primary antibodies used in the present study were anti-PD-L1 (catalog no. 29122, Cell Signaling Technology, Danvers, MA, USA 1:500), anti-HKII (catalog no. ab209847; Abcam, Cambridge, UK; 1:500), and anti-β-actin (Santa Cruz Biotechnology, catalog no. sc-47778; 1:5000). WB using biological replicates showed similar expression data, indicating the reproducibility of the results. For band quantification, images were analyzed using ImageJ software39 (link).
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