M7240

Initially, the paraffin sections were removed using xylene after warming for 1 h in a wet box. Subsequently, enzymatic digestion with hyaluronidase (18240-36; Nacalai Tesque, Inc., Kyoto, Japan) was performed to expose the antigens in the specimens. Additionally, to minimize non-specific reactions, the specimens were blocked using 3% bovine serum albumin. Then, the specimens were subjected to incubation with the following primary antibodies at 4 °C: anti-ki67 (× 200, M7240, Abcam, Cambridge, United Kingdom), anti-TNAP (× 300, ab65834, Genetex), anti-ENPP1 (× 300, rabbit, Genetex), anti-Col2 (× 300, MA5-12789, Thermo Fisher, Tokyo, Japan), and anti-Col10 (× 300, GTX37732, Genetex). Finally, the specimens were further incubated with secondary antibodies for 30 min at 25 °C and then mounted with a DAPI-containing mountant for fluorescence observation (Abcam). The histological analysis, specifically the counting of Ki67-positive cells, was performed on samples from three rats, with all positive cells counted through visual inspection. Furthermore, standardized exposure times of 250 ms for Ki67 and 3 ms for DAPI were applied, and the enumeration of positive cells was performed visually. For light observations, TNAP and ENPP1 were combined with diaminobenzidine.

HT-29 tumors were dissected at a size of about 1,000 mm³ and directly transferred to formalin. After fixation the tumors were embedded in paraffin and cut in 4 μm-sections. The sections were stained with hematoxylin and eosin (Biocare Medical) using a Tissue-Tek Prisma Plus, Sakura instrument according to the manufacturer’s instructions. Immunostaining with Anti-Ki67 (DAKO, M7240) and Anti-Annexin V (abcam, ab108321, rabbit) was performed after pretreatment with Targen Retireval Solution, Low pH, Dako, K800521-2; antibody dilution 1: 1000, 30 min RT and cMet (Abcam, ab51067), pretreatment – Target Retrieval Solution, High pH, Dako, K800421-2; antibody dilution 1: 100, overnight incubation then detected with Dako EnVision Flex High pH # K801021-2 kit.
Necrotic areas were quantified by estimating the fraction of necrosis in the whole tumor area in five 10× power fields. Mitotic and apoptotic cells (Annexin V positive) were scored in ten 40× power fields. cMet expression scored according to staining intensity (negative -, weak+, moderate++, or strong+++). The proliferation index was quantified by calculating the fraction of Ki67 positive cells in an 0.25 mm² grid in a hot spot area. All histopathologic evaluation was done in a blinded manner by a pathologist.