Mouse monoclonal cd4

Four micron thick sections of formalin-fixed, paraffin-embedded tissue underwent heat-induced epitope retrieval using CC1 (Ventana Medical Systems, Inc., Tucson, Arizona, USA), a Tris-based buffer at pH 8–8.5, followed by IHC staining with rabbit monoclonal CD3 (Ventana), mouse monoclonal CD4 (Leica Biosystems, Ltd., Newcastle, United Kingdom), rabbit monoclonal CD8 (Ventana), mouse monoclonal CD20 (Ventana), mouse monoclonal CD68 (Dako, Carpinteria, CA, USA), mouse monoclonal CD138 (Dako), mouse monoclonal HLA-II (anti-HLA-DP, -DQ, -DR) (Dako), or mouse monoclonal C4d (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Four-micron-thick sections of formalin-fixed, paraffin-embedded tissue were subjected to heat-induced epitope retrieval using CC1 (Ventana Medical Systems Inc., Tucson, Arizona, USA), a Tris-based buffer at pH 8–8.5, and immunohistochemistry staining. We used rabbit monoclonal CD3 (Ventana Medical Systems, Inc.), mouse monoclonal CD4 (Leica Biosystems Ltd, Newcastle, UK), rabbit monoclonal CD8 (Ventana Medical Systems, Inc.), mouse monoclonal CD20 (Ventana Medical Systems, Inc.), mouse monoclonal CD68 (Dako, Carpinteria, California, USA), mouse monoclonal CD56 (Dako), and mouse-monoclonal CD117 (Dako). The slides were scanned for each case using the Aperio ScanScope (Aperio Technologies, Vista, CA, USA). After scanning the slides, relevant areas of tumor were selected for analysis. Aperio membrane IHC algorithm was used for each marker, which is based on the spectral differentiation between brown (positive) and blue (counter) staining. Results of Aperio IHC quantification were also confirmed manually. We calculated the proportion of positive cells out of all cells in the sampled areas. The final results were validated by another pathologist, M.C.