The levels of TNF-α and IL-1β (Novus Biologicals, Inc.) in pleural effusions and the blood of mice were determined. Anti-CD8 antibody (Abcam), anti-PD1 antibody (EPR20665, Abcam), anti-PDL1 antibody (EPR20529, Abcam), anti-GAPDH antibody (ab8245, Abcam), and anti-β-actin antibody (13E5, CST) were used for antigen detection. Lymphocyte and tumor cell lines were co-cultured in 96-well plates at a ratio of 20:1. The LDH release experiment was then performed using the lactate dehydrogenase cytotoxicity test kit (Beyotime Biotechnology).
Epr20665
Manufactured by Abcam
Sourced in United Kingdom, United States
The EPR20665 is a laboratory equipment product. It serves as a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Hematoxylin, by Vector Laboratories (2 mentions)
Fjk 16, by Thermo Fisher Scientific (2 mentions)
Dab substrate, by Cell Signaling Technology (2 mentions)
Biotinylated anti rabbit or anti rat secondary antibodies, by Cell Signaling Technology (2 mentions)
F4 80, by Abcam (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Lactate dehydrogenase cytotoxicity test kit, by Beyotime (1 mentions)
Tnf α, by Novus Biologicals (1 mentions)
Il 1β, by Novus Biologicals (1 mentions)
Ab8245, by Abcam (1 mentions)
Epr21769, by Abcam (1 mentions)
Protein block, by Agilent Technologies (1 mentions)
Cal49, by Abcam (1 mentions)
Immunoglobulin g igg, by Abcam (1 mentions)
Ripa lysis buffer, by Abcam (1 mentions)
Ep1580y, by Abcam (1 mentions)
Vectra polaris automated quantitative pathology imaging system, by Akoya Biosciences (1 mentions)
Opal polaris 7 color manual ihc kit, by Akoya Biosciences (1 mentions)
Phenochart software version 1, by Akoya Biosciences (1 mentions)
D2s9r, by Cell Signaling Technology (1 mentions)
7 protocols using epr20665
1
Cytokine Levels and Lymphocyte Cytotoxicity
2
Assessing Tumor Immune Landscape
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To evaluate the changes in immunogenic microenvironment in treated tumors, we collected tumors seven days after the last treatment and incubated paraformaldehyde-fixed tumor tissue sections with primary antibodies (CD8, PD1) for 2 h at 37 °C and then washed them with PBS three times. CD8 (EPR21769, Abcam) and PD1 (EPR20665, Abcam) were purchased from Abcam company. Image-Pro Plus 6.0 was adopted to analyze each, and mean density (IOD/area) was used to analyze protein expression.
3
Immunohistochemical Analysis of Tumor Microenvironment
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One-third of the tumor, spleen, and one mammary were removed from female MMTV-Neu and PyMT mice and fixed in 10% phosphate-buffered formalin for 48 h, embedded in paraffin blocks, and sectioned (5–6 µm). Hydrogen peroxide was used to quench endogenous peroxidase activity. Sections were immunostained with antibodies raised against CD206 (1:200, Abcam), Gr1 (1:40, BM8, R&D), CD8 (1:40, 53–6.7, Biolegend), FOXP3 (1:50, FJK-16s, EBiosciences), PD-1 (1:100, EPR20665, Abcam), PD-L1 (1:50, MIH6, Abcam; R&D 1:50 for dual staining), cleaved caspase 3 (1:100, 5A1E, Cell Signaling), PCNA (1:200, sc-56, Santa Cruz Biothechonlogies), p-STAT1 (1:50, Cell Signaling), F4/80 (1:50, Abcam) and visualized with biotinylated anti-rabbit or anti-rat secondary antibodies (Cell Signaling or Vector Labs). Signal was detected using a DAB substrate (Cell Signaling) following the manufacturer’s recommendations. Sections were counterstained with hematoxylin (Vector Labs). Dual staining was performed with a Double Stain IHC kit using AP-rabbit and HRP-rat antibodies (Abcam, ab183285).
4
Quantifying PD-1 Expression in RSV-Immunized Cotton Rats
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Half the lungs from PBS, RSV, FI-RSV and FI-Mock immunized cotton rats were fixed in 10% formalin 5 days following RSV challenge, trimmed, processed and embedded into paraffin blocks. Charged slides were made from the blocks and used for immunostaining as previously described64 ,65 (link) with some modifications. Following 10 min antigen retrieval by boiling in 1 mM Tris/EDTA buffer (pH = 9.0) in a pressure cooker, sections were blocked with Protein Block (X0909, DAKO) for 2 h, and incubated overnight at 4 °C with rabbit anti-mouse PD-1 (Abcam, clone EPR20665). Then, the samples were incubated with a secondary anti-rabbit IgG followed by addition of diaminobenzidine (DAB) substrate.
For each section, 8–10 fields of view were counted at 20X magnification. Counting was performed using Northern Eclipse software and thresholds were set using negative controls. Fold change of percent PD-1 positive cells in the immunized groups over unimmunized control groups is presented.
For each section, 8–10 fields of view were counted at 20X magnification. Counting was performed using Northern Eclipse software and thresholds were set using negative controls. Fold change of percent PD-1 positive cells in the immunized groups over unimmunized control groups is presented.
5
Protein Expression and Western Blotting
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Total protein was extracted from both cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Abcam, UK). Protein concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA). An equal amount of protein from both cells was separated using 12% SDS–polyacrylamide gel and transferred onto a nitrocellulose membrane. The blotted membranes were blocked with Tris-buffered saline containing 5% non-fat milk to avoid nonspecific binding sites before the addition of primary antibodies i.e., anti-PD1 (EPR20665, 1:1000, Abcam, UK), anti-CTLA4 (CAL49, 1:1000, Abcam, UK), and anti-beta-actin (EPR21241, 1:5000, Abcam, UK). Beta-actin served as an internal control. After 24 h of incubation at 4 °C, the membrane was incubated again for 1 h with secondary antibody i.e., Immunoglobulin G (IgG) (1:100,000, Abcam, UK) at room temperature. Finally, the proteins were detected using Enhanced Chemiluminescence (ECL) Substrate Kit (Abcam, UK).
6
Multiplex IHC for Immune Profiling
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Multiplex immunohistochemical staining was performed following the protocol of the Opal Polaris 7-Color Manual IHC Kit (Akoya Bioscience, NEL861001KT). Antibodies against mouse CD8α (1:800, D4W2Z), F4/80 (1:200, D2S9R) and GFP (1:400, D5.1) were purchased from Cell Signaling Technology. Antibodies against mouse PD-1 (1:100, EPR20665), and CK19 (1:1500, EP1580Y) were purchased from Abcam. Slides were imaged, whole slides were scanned using a Vectra Polaris Automated Quantitative Pathology Imaging System (Akoya Biosciences), and multispectral images were acquired using Phenochart software, version 1.0.12 (Akoya Biosciences), to unmix and remove autofluorescence. The distances between CD8+ cells andCK19+ cells were calculated by HALO software (Indica Labs).
7
Immunohistochemical Analysis of Tumor and Lung
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A third of the tumor from MMTV-neu or the left lung from A/J mice were fixed in NBF for 48 h, embedded in paraffin, and sectioned. Endogenous peroxidase was quenched by hydrogen peroxide. Sections were immunostained with the following antibodies: CD206 (1:200, Abcam, Cambridge, United Kingdom), CD4 (1:40, GK1.5, Biolegend, San Diego, CA, USA), CD8 (1:40, 53–6.7, Biolegend, San Diego, CA, USA), FOXP3 (1:50, FJK-16s, EBiosciences, San Diego, CA, USA), PD-1 (1:100, EPR20665, Abcam, San Diego, CA, USA), p-ERK (1:100, 5A1E, Cell Signaling, Danvers, MA, USA), PCNA (1:200, sc-56, Santa Cruz Biotechnologies, Dallas, TX, USA), and F4/80 (1:50, Abcam, Cambridge, United Kingdom) and visualized with biotinylated anti-rabbit or anti-rat secondary antibodies (Cell Signaling or Vector Labs). Signal was detected using a DAB substrate (Cell Signaling) following the manufacturer’s recommendations. Sections were counterstained with hematoxylin (Vector Labs).
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