Anti human cd20cy clone l26

PLP-fixed frozen samples were cut in a cryostat at −22 °C into 5-μm sections and mounted on PEN-Membrane slides (Leica, Wetzlar, Germany). The tissue sections were stained with mouse anti-human PD1 (NAT105 ab52587, Abcam, Cambridge, UK) and anti-human CD20cy (clone L26, Dako, Michigan, MI, USA) antibodies, diluted 1:2000 and 1:1000, respectively, and detected by use of the Envision⁺ Dual Link System-HRP (Dako). The tissue sections were then counterstained with hematoxylin (Mayer's hematoxylin, Muto Pure Chemical, Tokyo, Japan) for 20 s at room temperature. After staining, tissue sections were dehydrated with ethanol and dried at room temperature before laser microdissection (LMD).

All paraffin-embedded organs were stained with H&E to perform a complete histopathological analysis and a clinical pathologist supervised all samples for toxicity evaluation. To detect the presence of human B-cells and to quantitate the percentage of organ infiltration, we used immunohistochemistry (IHC) staining with an antibody anti-human CD20cy (clone L26, Dako). Firstly, 5 and 27 low power fields at 100 magnifications were taken for LNs and BM, respectively, in each mice group (buffer or T22-PE24- H6). Secondly, the area occupied by B cells in each organ was selected and the percentage of CD20+ cells was quantified using the cellSens Dimension 1.9 software (Olympus).
IHC staining was performed in a DAKO Autostainer Link48 (Agilent) following the manufacturer's instructions. Representative pictures were taken using an Olympus DP73 digital camera and processed with the cellSens Dimension 1.9 software (Olympus) at 200 or 400 magnifications.