The tissue samples were fixed in formalin and embedded in paraffin, and the paraffin blocks were sliced into 4 µm sections to conduct immunohistochemical (IHC) staining. Six serial sections of each paraffin-embedded tumor block were cut, one for H&E staining and five for IHC staining. The primary antibodies for TIL subsets were mouse monoclonal anti-human CD8 (DakoCytomation, Glostrup, Denmark; 1 : 100 dilution), granzyme B (Novocastra, Newcastle, UK; 1 : 100), OX40 (Novocastra; 1 : 30), FOXP3 (Abcam, Cambridge, UK; 1 : 50). The identification for the TLSs was the primary antibody to MECA-79 (1 : 300 dilution, Santa Cruz, California, USA), which recognizes sulfate-dependent carbohydrate epitopes of peripheral node addressin (PNAd) expressed in endothelial cells of HEVs. The IHC was performed as recommended by the manufacturer. The antigen retrieval was done by heat-induced epitope retrieval methods for 1 min and 30 s in citric buffer (pH 6.0). The detailed steps were the same as in our previous study [26 (link)]. Dako EnVision (DakoCytomation, Denmark) was used as the secondary antibody. Negative controls were done by omitting the primary antibodies. The tonsil tissue was used as positive controls.
Mouse monoclonal anti human cd8
Manufactured by Agilent Technologies
Sourced in Denmark
Mouse monoclonal anti-human CD8 is a laboratory equipment product that detects the CD8 protein found on the surface of certain T cells. It can be used in various immunological and cell biology applications.
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Lab products found in correlation
2 protocols using mouse monoclonal anti human cd8
1
Immunohistochemical Analysis of Tumor-Infiltrating Lymphocytes
2
Multimodal Biomarker Evaluation for Cancer
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Primary endpoints will be assessed centrally for all trial sites when the number of participants required for an interim or final analysis has been reached.
All pseudo-anonymised imaging data will be transferred to the trial radiologist for the measurement of the tissue transfer constant Ktrans on DCE-MRI. Following motion correction, the MiStar software (Apollo Medical, Melbourne, Australia) will be used for kinetic modelling of the contrast-enhancement data using the extended Tofts model [42 (link)] and a model arterial input function [43 (link)]. Regions of interest encompassing the entire tumour will be defined and independently reviewed by two radiologists. Two radiologists verify the correct recording of the median Ktrans value independently.
The CD8+ T-cell count will be established centrally following transfer of the formalin-fixed paraffin-embedded tissue blocks. Following slicing of the block, endogenous peroxidase suppression and heat-mediated antigen retrieval, the mouse monoclonal anti-human CD8 (DAKO Cytomation, Glostrup, Denmark, 1:100 dilution) will be the primary antibody. Slides will be scanned and two board-certified uro-pathologists will quantify the CD8+ T-cells in ten random fields of view per tumour sample, expressed as the median number of cells/mm2 before and after treatment.
All pseudo-anonymised imaging data will be transferred to the trial radiologist for the measurement of the tissue transfer constant Ktrans on DCE-MRI. Following motion correction, the MiStar software (Apollo Medical, Melbourne, Australia) will be used for kinetic modelling of the contrast-enhancement data using the extended Tofts model [42 (link)] and a model arterial input function [43 (link)]. Regions of interest encompassing the entire tumour will be defined and independently reviewed by two radiologists. Two radiologists verify the correct recording of the median Ktrans value independently.
The CD8+ T-cell count will be established centrally following transfer of the formalin-fixed paraffin-embedded tissue blocks. Following slicing of the block, endogenous peroxidase suppression and heat-mediated antigen retrieval, the mouse monoclonal anti-human CD8 (DAKO Cytomation, Glostrup, Denmark, 1:100 dilution) will be the primary antibody. Slides will be scanned and two board-certified uro-pathologists will quantify the CD8+ T-cells in ten random fields of view per tumour sample, expressed as the median number of cells/mm2 before and after treatment.
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