The single-plex chromogenic IHC was performed as published (24 (link)-31 (link)) (Figure 1 ). Ten primary antibodies for KIR 2D (L1, L3, L4, S4) (BC032422/ADQ31987/NP_002246/NP036446, 1/75; Abcam, Cambridge, MA, USA), KIR 3D (L1) (AA 1-444, 1/1,500; Abcam), MHC Class II DP DQ DR (CR3/43, 1/100; Abcam), PD-1 (NAT 105, predilute; Cell Marque, Rocklin, CA, USA), PD-L1 (22C3; Dako, Carpenteria, CA, USA), GAL-9 (NBP2-45619; Novusbio, CO, USA), OX40, OX40L, LAG-3, and TIM3 (EPR4392, 1/1,000; Abcam) were applied on TMA slides from the internal cohort. The antibodies for PD-1 (ZM-0381, 1/100, Golden bridge zhongshan, Beijing, China), PD-L1 (13684S, 1/300, Cell Signaling, Beverly, MA, USA) were performed on whole IHC slides from the external cohort.
Epr4392
Manufactured by Abcam
Sourced in United States
EPR4392 is a recombinant human monoclonal antibody that recognizes the protein Synaptopodin. It is intended for use in research applications.
Automatically generated - may contain errors
4 protocols using epr4392
1
Multiplex Chromogenic IHC for Immune Checkpoint Markers
2
Evaluating Tumor Immune Landscape via IHC
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Based on the results of the NanoString assay, we performed immunohistochemistry (IHC) for LAG‐3 (clone EPR4392; Abcam) and PD‐L1 (clone SP142; Ventana). All slides were scanned using a Pannoramic 1000 digital slide scanner (3DHistech). An INFINITT DPS (INFINITT Healthcare) was used as the image viewing system. Images with an area of 0.37 mm2 were captured for two representative areas to measure the degree of infiltration of LAG‐3‐positive cells in tumors. The number of positive cells and the ratio (%) of positive cells to total cells in the captured images were investigated using QuPath software.23 A combined positive score (CPS) (the number of positive tumor cells, lymphocytes and macrophages divided by the total number of viable tumor cells, multiplied by 100) system was used to evaluate PD‐L1 expression. One pathology resident (HL) and one hematopathologist (JC) independently measured the CPS of all PD‐L1 stains, and the scores of cases with discrepancies were established by consensus.
3
Immunohistochemical Profiling of TNBC
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Formalin-fixed and paraffin-embedded sections (4 µm) from TNBC samples were used for immunohistochemistry (IHC) staining. Briefly, these sections were deparaffinized with xylene and rehydrated with a graded ethanol series (100%, 95%, and 70%) and distilled water according to standard immunohistochemical protocols. After rehydration, heat-induced antigen retrieval was performed using citrate buffer (pH=6.0) for 1.5 minutes in a water bath. Tissue sections were then soaked in a 3% H2O2 solution for 30 minutes. Rabbit monoclonal LAG-3 (1:1,000, EPR4392; Abcam, Cambridge, USA), PD-1 (1:200, EPR4877; Abcam), PD-L1 (1:200, EPR19759; Abcam), or CD8 (1:100, ab4055; Abcam) antibody was applied at 4℃ overnight. Each section was incubated with secondary antibody at 37℃ for 20 minutes, then visualized by diaminobenzidine for 3 to 10 minutes and counterstained with hematoxylin. For the negative control samples, the primary antibody was substituted with phosphate-buffered saline. Positive control samples were developed according to the manufacturer's instructions for each antibody.
4
Immunohistochemical Analysis of Tumor Biomarkers
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Tumor tissue was obtained from each patient at the time of diagnosis, acquired from surgical specimens and small biopsy specimens through percutaneous needle biopsy, bronchoscopic biopsy, and endoscopic bronchial ultrasonography biopsy. Specimens were fixed with formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E). Additional IHC staining was carried out to determine tumor NY‐ESO‐1, LAG‐3, and PD‐L1 expression. The slides were treated according to standard protocol, fixed in neutral buffered formaldehyde, and processed into paraffin wax, and then incubated overnight with primary antibodies against NY‐ESO‐1 (E978, Invitrogen), LAG‐3 (EPR4392, Abcam), and PD‐L1 (22C3, Dako). Absence or presence of NY‐ESO‐1 expression on tumor cells and LAG‐3 on immune cells were evaluated by an experienced lung cancer pathologist (Professor JH Chung). Semiquantitative assessment was performed by estimating the percentage of positive cytoplasmic staining (Fig S1). To predict progression‐free survival (PFS) and overall survival (OS), a >5% cutoff was used for LAG‐3 and NY‐ESO‐1, while 5% and 50% cutoffs were used to analyze PD‐L1 expression.
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