Anti active caspase 3 antibody

Proliferation, cell death and autophagic processes in xenografts were determined by immunohistochemistry for Ki67 marker using an anti-Ki67 antibody (Sigma-Aldrich), for active caspase-3 marker using an anti-active caspase-3 antibody (R&D Systems) and for LC3b-II marker using an anti-LC3b-II antibody (Cell Signaling), respectively. Hematoxylin and Eosin (H&E) staining was also performed. For quantification, Ki67 positively stained cells in six consecutive and independent fields were counted from the edge towards the center of each section. Photographs for quantification were taken with a Leica DM4000B microscope (Leica).

For antigen retrieval, paraffin sections were digested by the same method with ISEL for MPO, or using 10 mM sodium citrate buffer for active caspase-3, sFGL2 and FcγRIIB. The sections were then blocked by peroxidase-blocking reagent and labelled by anti-MPO (1:600 dilution, DAKO), anti-active caspase-3 antibody (1:100 dilution, R&D System, Minnesota, USA), anti-sFGL2 (1:200 dilution, Abcam, Cambridge, UK) or anti-FcγRIIB (1:200 dilution, Abcam) antibodies at 4°C overnight. The antibody binding was revealed by AEC for MPO and active caspase-3, and DAB for sFGL2 and FcγRIIB. MPO + cells and active caspase-3+ cells in the renal cortex were manually counted, while the expression of sFGL2 and FcγRIIB were semi-quantitatively scored using optical volume density (OD × mm²) analysis (Image-Pro Plus 6.0, Media Cybernetics Inc., Bethesda, USA) in 20 fields at 400× magnification.