Cd8 clone sp57

Serial 3-µm sections were prepared from one representative formalin-fixed paraffin-embedded (FFPE) block. IHC staining was performed in all GCLS cases with antibodies against cytokeratin (CK) AE1/AE3 (clone PCK26, prediluted; Ventana Medical Systems), CD3 (clone LN10, 1:500; Leica Biosystems), CD8 (clone SP57, prediluted; Ventana Medical Systems) and PD-L1 (clone E1L3N, 1:1000; Cell Signaling Technology) [17, (link)34, (link)35] (link). Samples were processed in the automatic Ventana Benchmark Ultra platform using the OptiView Universal DAB detection kit and the OptiView Amplification kit for PD-L1 staining. EBV infection was studied by chromogenic ISH for EBV-encoded RNA (EBER-ISH, INFORM EBER probe, Ventana Medical Systems) using the same equipment, with enzymatic digestion (ISH protease) and the iViewBlue detection kit. Two TMA 3 µm sections were prepared and used for PD-L1 IHC and EBER-ISH.

Three μm-thick sections were cut from 1 core formalin-fixed and paraffin-embedded tissue of a 16-gauge needle biopsy and stained using antibodies against CD3 (clone 2GV6, Roche), CD4 (clone SP35, Roche), CD8 (clone SP57, Roche), CD20 (clone L26, Roche), CD68 (clone PGM1, Diagnostic Biosystems), CD56 (clone MRQ42, Roche), CD138 (clone B-A38, Roche) and TIA1 (2G9A10F5, Vitro Máster Diagnóstica). Here, TIA1 marker was used as a surrogate marker of cytotoxic T and NK cell granules. 39 39. Tian, Q. Immunostains were automatically performed with a Benchmark XT (Roche) and revealed with DAB Optiview Kit (clone B-A38, Roche). Stained inflammatory cells were quantitatively assessed by 2 pathologists blinded to clinical background under light microscopy in the glomerular capillaries, peritubular capillaries, and interstitial compartment, especially when forming aggregates in scarred areas. A standardized evaluation of the cell count in renal parenchyma was conducted according to renal compartments. In glomerular capillaries, cell count was standardized by dividing the number of cells by the total glomeruli. In peritubular capillaries and renal interstitial compartments, cell count was standardized by dividing the number of cells by the length of the sample in millimeters.

All biopsy specimens were fixed in 10% buffered formalin and embedded in paraffin. Unstained slides for immunohistochemistry were available in five nivolumab-treated cases and all cases of autoimmune hepatitis or druginduced liver injury. Immunostaining was performed on a Ventana Benchmark XT (Ventana Medical Systems, Inc., Tucson, AZ) or Bond Max autostainer (Leica Microsystems, Wetzlar, Germany) according to the manufacturers' protocols. Deparaffinized sections were heattreated and incubated with primary antibodies. Immunohistochemical staining for multiple lymphocyte markers was performed using the following antibodies: CD3 (clone F7.2.38, 1:50, Dako Cytomation, Glostrup, Denmark), CD4 (clone 1F6, prediluted, Leica Microsystems), CD8 (clone SP57, Roche, Ltd., Basel, Switzerland), and CD20 (clone L26, prediluted, Dako Cytomation). The numbers of lymphocytes positive for each marker were counted at three hot spots and calculated as the average per high power field. Ratios of CD20+ /CD3+ and CD4 +/CD8+ cells were also calculated in each case.