Anti cleaved caspase 3 monoclonal antibody

On day 1 and 3 after LI, PPU-7 cells on chamber slides were fixed with 4% paraformaldehyde for 15 min at room temperature and incubated in a blocking solution (1% BSA, 10% normal goat serum) for 1 h at room temperature. For primary antibody application, the dilution of anti-cleaved caspase-3 monoclonal antibody (Cell Signaling, Danvers, MA, USA) was used at 1:500, and the cells were incubated overnight at 4 °C. For secondary antibody application, diluted HRP-conjugated goat anti-rabbit IgG H&L antibody (abcam, Cambridge, UK) was used at 1:500, and the cells were incubated for 1 h at room temperature. The positive signal was detected using 3,3-diaminobenzidine (DAB) (TaKaRa, Kusatsu, Japan) as a staining substrate. Sections were counterstained to observe clear tissue and cell morphology using hematoxylin. Light micrographs were obtained using a Canon EOS Kiss X8i (Canon, Tokyo, Japan) camera on an optical microscope (OLYMPUS BX50, Olympus, Tokyo, Japan). The number of apoptotic cells present on a chamber slide expressed as a fraction of the total number of cells, named the “apoptotic index,” was used to evaluate apoptotic state. The number of activated caspase-3-labeled apoptotic cells and bodies was calculated in 30 high power fields (HPFs; objective X400, field diameter 640 μm). The apoptotic index was calculated as the percentage of the whole PPU-7 population.

Briefly, dissected tumor tissues were fixed in 10% neutral buffered formalin overnight and then embedded in paraffin and cut into 4μm-thick sections. The sections were deparaffinized with xylene and rehydrated with gradient alcohol before antigen retrieval was performed in 0.01 M citrate buffer at just below boiling temperature for 10 min. Endogenous peroxidase activity was quenched with 3% H₂O₂ and then 10% goat serum were used for eliminating non-specific binding. Subsequently, tissue sections were incubated overnight at 4 °C with anti-Ki-67 monoclonal antibody (cat. no. 14-5698-82; Thermo Fisher Scientific) or anti-cleaved-Caspase-3 monoclonal antibody (Cell Signaling Technology, MA, USA, cat. no. 9664;), at 1:1000 and 1:500 dilutions, respectively. Then the slides were washed and incubated with biotin-conjugated secondary antibodies for 30 min followed by incubation with ABC reagents in the Vectastain Elite ABC kits (Vector Laboratories, CA, USA, cat. no. PK-7200) and counterstaining with hematoxylin, as previously described [42 (link)]. Images were obtained using an Olympus microscope with DP Controller software (Olympus, Waltham, MA, USA, version 3.2.1.276).