Multi-color multiplex immunohistochemistry (IHC) with CD8 for cytotoxic T lymphocytes (clone MRQ26, mouse, Ventana), CD163 for macrophages (clone SP57, rabbit, Ventana), and PD-L1 (clone SP263, rabbit, Ventana) was performed on freshly cut whole sections from pretreatment biopsies as described before60 (link),61 (link). A membranous PD-L1 staining in tumor cells or immune cells was considered as specific staining. The immunohistochemistry was evaluated with consensus viewing by two pathologists (Y. Hou and Z. Li). The percentage of PD-L1 positively-stained cells were recorded and used for machine learning models (features that generated by pathologists). The parameters assessed were as follows: PD-L1 expression in tumor cells (PD-L1 TC), PD-L1 expression in immune cells (PD-L1 IC), PD1 expression in immune cells, intratumoral CD8+ immune cells (IT-CD8+), peritumoral CD8+ immune cells (PT-CD8+), intratumoral CD163+ macrophages (IT-CD163+), and peritumoral CD163+ macrophages (PT-CD163+).
Clone sp57
Manufactured by Roche
Sourced in United Kingdom, United States
The Roche Clone SP57 is a laboratory equipment designed for gene cloning and expression. It is a compact and efficient instrument that streamlines the cloning process, enabling researchers to perform DNA manipulations with precision and consistency. The Clone SP57 is engineered to provide reliable and reproducible results, making it a valuable tool for molecular biology and genetic research applications.
Automatically generated - may contain errors
4 protocols using clone sp57
1
Multiplex IHC for Tumor Immunophenotyping
2
Characterizing Immune Cell Populations in Tonsil and HL
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IHC was performed on 4µm serial FFPE tonsil and HL biopsy sections to characterize cell populations with the following antibodies: CD8 for cytotoxic T lymphocyte (CTL) (clone SP57, Ventana Roche), Granzyme B (GrB) for activated cytotoxic cells (clone GB7, AbDSerotec, Oxford, UK), CD68 for macrophages (clone KP-1, Ventana Roche), Foxp3 for regulatory T lymphocytes (Treg) (Abcam, Cambridge, UK), IL10 for anti-inflammatory cytokine productive cells (Abcam), and CD4 for T helper (Th) (Ventana Roche).
All cell markers were observed and counted by two pathologists in serial slides on the basis of the best-preserved areas around EBV+ and EBV− zones in both HL and tonsil samples. The results were expressed as immunopositive cells number/total cell number.
All cell markers were observed and counted by two pathologists in serial slides on the basis of the best-preserved areas around EBV+ and EBV− zones in both HL and tonsil samples. The results were expressed as immunopositive cells number/total cell number.
3
Quantifying Immune Cell Infiltration in Tumor Samples
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We performed immunohistochemical staining on paraffin-embedded sections (4 μm thick) of tumor specimens from the FUSCC cohort (n = 165) to evaluate the expression of CD4, CD8, and CD86. Sections were deparaffinized and rehydrated through a graded series of xylene-ethanol baths. Antigen retrieval was performed using a citrate buffer, pH 6.0, for 20 min at 95°C. Tissue sections were incubated with primary antibody for 1 h at room temperature. The antibodies were used in a 1:200 dilution for anti-CD4 (Clone EPR6855, Abcam), without dilution for anti-CD8 (clone SP57, Ventana), and 1:200 dilution for anti-CD86 (13395-1-AP, Proteintech). Visualization was performed using the Novolink Polymer Detection Systems (Leica Biosystems). Tissue sections were counterstained with hematoxylin, dehydrated, and mounted. The analysis was performed in a blinded fashion. Images were organized in folders identified by patient ID by one person and quantified by another. Results were recorded as percentage of IHC-stained cells and adjudicated by two pathologists.
4
Immunohistochemical Profiling of Tonsil Cells
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IHC was performed on 4 µm serial FFPE tonsil sections to characterize cell populations with the following antibodies: CD8 for cytotoxic T lymphocyte (CTL) (clone SP57, Ventana Roche, Tucson, USA), Granzyme B (GrB) for activated cytotoxic cells (clone GB7, AbD Serotec, Oxford, UK), CD68 for macrophages (clone KP-1, Ventana Roche), IL10 for anti-inflammatory cytokine-productive cells (Abcam), Foxp3 for regulatory T lymphocytes (Treg) (Abcam, Cambridge, UK), PD1 (CD279) for T cell-negative regulator (AbD Serotec), CD56 for NK cells (Leica, Buffalo, IL, USA) and CD4 for T helper (Th) (Ventana Roche). Lymph node reactive hyperplasia tissue was used as positive control. Negative controls for each case consisted in substituting the primary antibody with antibody dilution buffer and an isotype control. The stains were developed using diaminobenzidine (DAB).
Given the fact that we previously characterized viral antigen expression in lymphocytes at germinal center (GC), interfollicular (IF) and subepithelial (SubEp) regions, the same approach was used for microenvironment characterization at those three histological regions [19, (link)20] (link).
All cell markers were observed and counted by two pathologists in serial slides on the basis of the best-preserved areas around LMP1+ and LMP1-zones. The results were expressed as immunopositive cells nº/100 total cell nº.
Given the fact that we previously characterized viral antigen expression in lymphocytes at germinal center (GC), interfollicular (IF) and subepithelial (SubEp) regions, the same approach was used for microenvironment characterization at those three histological regions [19, (link)20] (link).
All cell markers were observed and counted by two pathologists in serial slides on the basis of the best-preserved areas around LMP1+ and LMP1-zones. The results were expressed as immunopositive cells nº/100 total cell nº.
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