Cd4 clone ep204

In-House RCC samples (n = 4) after surgery were fixed in 4% paraformaldehyde, embedded in paraffin and five-micrometer sections were used for the immunofluorescence staining. Multiplex IHC was performed using Opal 6-plex Detection Kit (cat: NEL821001KT, Akoya Biosciences, Menlo Park, USA). A multiplex panel of immune markers was developed with antibodies against CXCR3 (clone EPR25373-32, cat: ab288437, dilution 1:200, Abcam, Cambridge, MA, USA), CD4 (clone EP204, cat: 104R-26, dilution 1:50, Cell Marque), CD8 (clone C8/144B, cat: M710301-2, dilution 1:200, Dako/Agilent, Santa Clara, CA, USA), CD68 (clone PG-M1, cat: M087601-2, dilution 1:250, Dako/Agilent), cytokeratin (clone AE1/AE3, cat: MA5-13156, dilution 1:500, Thermo-Fisher). The staining procedure was performed using an automated staining system (BOND-RX; Leica Biosystems, Vienna, Austria). To visualize cell nuclei, the tissue was stained with 4’,6-diamidino-2-phenylindole (spectral DAPI, Akoya Biosciences). Slides were scanned at 20x magnification using Mantra 2 Quantitative Pathology Workstation (Akoya Biosciences) and representative images from each tissue were acquired with the Mantra Snap software version 1.0.4. Image spectral deconvolution, multispectral image analysis and cell phenotyping was carried out using the InForm Tissue Analysis Software version 2.4.10 (Akoya Biosciences).