Bz 8000 confocal microscope

Formalin-fixed and paraffin-embedded tumor sections were pretreated with citrate buffer for 40 min at 95°C, quenched with 0.05% H₂O₂, and incubated overnight at 4°C with the following primary antibodies in Can Get Signal Immunostain Solution A (Toyobo): rabbit anti-human VEGF polyclonal antibody (1∶50) (Abcam, Cambridge, UK), rabbit anti-human HIF-1α antibody (1∶50) (Cell Signaling Technology), mouse anti-human MMP-2 antibody (1∶100) (Novocastra Laboratories, New-castle, UK) and mouse anti-human MMP-9 antibody (1∶100) (Novocastra Laboratories). Following this treatment, sections were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG polyclonal antibody (Nichirei Bioscience, Tokyo, Japan) for 30 min at room temperature. Signals were developed as a brown reaction product using peroxidase substrate 3′,3′-diaminobenzidine (Nichirei Bioscience). The sections were counterstained with hematoxylin and examined with a BZ-8000 confocal microscope (Keyence, Osaka, Japan) [5] (link).

To induce myogenic differentiation, cells were rinsed thoroughly with PBS 24 h after siRNA transfection and then cultured with DMEM containing 2% horse serum (Thermo Fisher Scientific, Waltham, MA, USA), penicillin (100 U/ml), and streptomycin (10 mg/ml). Three days later, cells were observed with a BZ-8000 confocal microscope (Keyence, Osaka, Japan) to assess morphological changes. For immunofluorescence, cells on coverslips were fixed with absolute methanol, washed, and incubated with anti-myosin heavy chain (MHC) antibody (M4276, Sigma-Aldrich) for 1 h, rinsed with PBS, incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG (A-11001, Invitrogen) for 1 h, and visualized using a fluorescence microscope as described previously [18 (link)].

Deparaffinized sections were digested with proteinase (Dako Retrieval Solution Ready-to-Use) for 20 min and treated overnight at 4°C with the following antibodies in Can Get Signal immunostain solution A: Rabbit anti-mouse p27 polyclonal antibody (1:50 dilution; Santa Cruz Biotechnology, Inc.). The secondary antibodies used were goat anti-rabbit immunoglobulin Alexa Fluor 488 (1:200 dilution; Life Technologies, Carlsbad, CA, USA) for 30 min at room temperature. The nuclei were stained with DAPI and images were captured using a BZ-8000 confocal microscope (Keyence).