Bz 9000 microscopy

Frozen tissue sections (8 μm thick) of tumours were fixed with 4% paraformaldehyde solution in PBS. To detect endothelial cells, rat anti-CD31/PECAM-1 monoclonal antibody (1:100 dilution, MEC 13.3, BD Pharmingen, Franklin Lakes, NJ) was used. To determine the hypoxia of tumour tissue, rabbit anti-CA9 polyclonal antibody (1:1,000 dilution, Novus Biologicals, Littleton, CO) was used. Appropriate secondary antibodies conjugated with peroxidase (ready to use, Nichirei, Tokyo, Japan) and the 3,3′-diaminobenzidine tetrahydrochloride (DAB) Liquid System (DAKO, Carpinteria, CA) were used to detect the immunostaining. Nuclei were counterstained with hematoxylin (DAKO). MVD (the number of CD31⁺ structures per HPF) was evaluated at a × 200 magnification15 (link)53 (link)66 (link). The CA9-positive areas were calculated using the Image J software program (National Institutes of Health, Bethesda, MD) at a × 200 magnification. Images were acquired by Keyence BZ-9000 microscopy (Keyence, Tokyo, Japan).

C26-conditioned medium (C26-CM) was prepared by culturing confluent C26 cells in serum-free DMEM for 48 h. The C26-CM medium was centrifuged (5000× g, 4 °C, 5 min) to remove debris. Then, C26-CM was diluted with DMEM supplemented with 2% FBS and added simultaneously with recombinant adiponectin to C2C12 myotubes to induce muscle atrophy-related reactions. At 48 h after the addition of C26-CM and adiponectin, C2C12 was lysed using sample buffer, and protein expression was analyzed. For morphological analysis of the myotubes, cells were fixed in 4% paraformaldehyde in PBS for 10 min, followed by treatment with PBS containing 0.1% TritonX-100 (Sigma-Aldrich). The cells were then stained with anti-MYH4 (1:2000, Thermo Fisher Scientific, MF20) and Hoechst 33342 (Sigma-Aldrich). Cell images were taken using BZ-9000 microscopy (KEYENCE) and were analyzed using ImageJ software (NIH, Bethesda, MD, USA).
A nonisotopic-labeled protein synthesis assay (SUnSET method) was performed as described previously [39 (link)]. In brief, puromycin (0.04 μM) was added to the culture medium 60 min before harvesting the cells. Protein was extracted and analyzed as stated in Section 4.8. Total protein was assessed with Coomassie blue staining, and puromycin-incorporated newly synthesized protein was detected by western blotting using a puromycin antibody (1:2000, CosmoBio).