Cells were pelleted, washed with PBS, and then lysed with Radio Immunoprecipitation Assay Lysis (RIPA) buffer (Thermo Scientific) supplemented with protease inhibitor cocktail (CST). Protein concentration was determined by the BCA Protein assay kit (Thermo Scientific) and lysates were then boiled for 10 min. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Pall). Membranes were blocked for 1 h at room temperature with 5% milk in 1 × Phosphate Buffered Saline Tween-20 (PBST) and incubated with the indicated primary antibodies at 4 °C overnight. After washing with PBST three times for 30 min, membranes were incubated with secondary antibodies at room temperature for 1 h. The membranes were washed with PBST three times and visualized with chemiluminescence (CLiNX). Antibodies against the following human proteins were used: GAPDH (SCB), PD-L1 (Yurogen), PD-L1 E1L3N (CST), and PD-L1 28-8 (Abcam). Band intensities were quantified by ImageJ and were normalized using GAPDH as a housekeeping protein. Results are representative of three independent experiments.
Pd l1 28 8
Manufactured by Abcam
Sourced in United States, United Kingdom
The PD-L1 28-8 is an antibody product used for the detection of the Programmed Death-Ligand 1 (PD-L1) protein. The PD-L1 28-8 antibody is designed to specifically bind to the PD-L1 protein, which plays a role in the regulation of the immune response.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Pd l1 e1l3n, by Abcam (1 mentions)
Pd l1, by Abcam (1 mentions)
P mertk ab192649, by Abcam (1 mentions)
Pd l1 e1l3n, by Cell Signaling Technology (1 mentions)
Cd8 c8 144b, by Agilent Technologies (1 mentions)
3 3 diaminobenzidine tetrahydrochloride, by Dojindo Laboratories (1 mentions)
Histofine simple stain max po multi kit, by Nichirei Biosciences (1 mentions)
Ki 67 mib 1, by Agilent Technologies (1 mentions)
Glut1 ab15309, by Abcam (1 mentions)
3 protocols using pd l1 28 8
1
Western Blot Analysis of PD-L1 Protein
2
Immunohistochemistry Analysis of PD-L1 and Related Markers
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The detailed procedures for H&E and IHC analysis were conducted as described in our previous study [33 (link)] and also mentioned in our supplementary materials. The antibodies used in our current study were as follows: PD-L1 (2B11D11, ProteinTech Group Inc., Chicago, IL, USA), PD-L1 (28-8, Abcam, Cambridge, UK), PD-L1 (E1L3N, Cell Signaling Technology, Danvers, MA), p-MerTK (ab192649, Abcam, Cambridge, UK) and Ki67 antibody (8D5, Cell Signaling Technology, Danvers, MA).
3
Immunohistochemical Staining Protocol
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We cut a 4-μm section from paraffin blocks of samples, thereafter mounting each section on a silane-coated glass slide, deparaffinising and soaking for 30 min at room temperature in 0.3% H2O2/methanol to block endogenous peroxidases. Supplementary Table 1 details the staining procedure.
Next, non-specific binding sites were blocked by incubating with 0.25% casein/1% BSA for 30 min at room temperature. The antibodies of PD-L1 (28-8; 1:400 dilution; Abcam, Cambridge, UK), CD8 (C8/144B; 1:100 dilution; DAKO, Glostrup, Denmark), HIF1-A (EP1215Y; 1:200 dilution; Abcam, Cambridge, UK), GLUT1 (ab15309; 1:200 dilution; Abcam, Cambridge, UK), E-cadherin (HECD-1; 1:500 dilution; Takara BIO, Shiga, Japan), and Ki-67 (MIB-1; 1:40 dilution; DAKO, Glostrup, Denmark) were visualised using the HRP/DAB (Polymer) Kit (Nichirei, Tokyo, Japan) and Histofine Simple Stain MAX-PO (Multi) Kit (Nichirei) per the manufacturer’s instructions. We applied the chromogen 3,3-diaminobenzidine tetrahydrochloride (Dojindo Laboratories, Kumamoto, Japan) as a 0.02% solution containing 0.005% H2O2 in 50-mM Tris–HCl buffer (pH 7.6). Thereafter, the sections were lightly counterstained with Mayer’s haematoxylin and mounted. Finally, we established negative controls by omitting the primary antibody.
Next, non-specific binding sites were blocked by incubating with 0.25% casein/1% BSA for 30 min at room temperature. The antibodies of PD-L1 (28-8; 1:400 dilution; Abcam, Cambridge, UK), CD8 (C8/144B; 1:100 dilution; DAKO, Glostrup, Denmark), HIF1-A (EP1215Y; 1:200 dilution; Abcam, Cambridge, UK), GLUT1 (ab15309; 1:200 dilution; Abcam, Cambridge, UK), E-cadherin (HECD-1; 1:500 dilution; Takara BIO, Shiga, Japan), and Ki-67 (MIB-1; 1:40 dilution; DAKO, Glostrup, Denmark) were visualised using the HRP/DAB (Polymer) Kit (Nichirei, Tokyo, Japan) and Histofine Simple Stain MAX-PO (Multi) Kit (Nichirei) per the manufacturer’s instructions. We applied the chromogen 3,3-diaminobenzidine tetrahydrochloride (Dojindo Laboratories, Kumamoto, Japan) as a 0.02% solution containing 0.005% H2O2 in 50-mM Tris–HCl buffer (pH 7.6). Thereafter, the sections were lightly counterstained with Mayer’s haematoxylin and mounted. Finally, we established negative controls by omitting the primary antibody.
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