Myo/Nog cells were identified in tissue sections by double labeling with the anti-BAI1 G8 IgM mouse mAb30 (link) and the anti-noggin goat polyclonal anti-serum (AF719; R&D Systems, Minneapolis, MN, USA), as described previously.25 (link),30 (link) Double labeling also was carried out with the IgM BAI1 mAb and IgG mAb antibodies to α-SMA (F3777 diluted 1:250; Sigma-Aldrich, St. Louis, MO, USA), striated muscle myosin II (ab58899 diluted 1:200; Abcam, Cambridge, MA, USA), the leukocyte markers CD68 (ab201340 diluted 1:200; Abcam), CD45 (ab10558 diluted 1:200; Abcam), and CD18 (MA1819 diluted 1:100; ThermoFisher Scientific, Waltham, PA, USA), and human nucleoli (ab190710 diluted 1:100; Abcam). The α-SMA mAb was directly conjugated with fluorescein. Other primary antibodies were visualized with AffiniPure Fab fragment subclass and species-specific secondary antibodies conjugated with Rhodamine Red or Alexa 488 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The level of background fluorescence was determined by incubating sections in secondary antibody only. Apoptotic cells were identified with fluorescent terminal deoxynucleotidyl transferase–dUTP nick-end labeling (TUNEL) reagents (Roche Diagnostics, Mannheim, Germany). Coverslips were applied with Dapi Fluoro-Gel II Mounting Medium (Electron Microscopy Sciences, Hatfield, PA, USA).
Tissues were analyzed with the Nikon Eclipse E800 epifluorescence microscope (Nikon Instruments Inc., Melville, NY, USA) equipped with 10 x, 60 x, and 100 x lenses, the Infinity 3S camera (Teledyne DALSA, Waterloo, Ontario, Canada) and Image Pro Plus image analysis software program (Media Cybernetics, Rockville, MD, USA) and the Olympus Confocal Fluoview 1000 microscope equipped with a 60 x oil immersion lens and Fluoview software program (Olympus Corp., Tokyo, Japan). Adobe Photoshop version 23 (Adobe Inc., San Jose, CA, USA) was used to adjust photographs for brightness and contrast, and assembly and annotation of figures.
Tissues were analyzed with the Nikon Eclipse E800 epifluorescence microscope (Nikon Instruments Inc., Melville, NY, USA) equipped with 10 x, 60 x, and 100 x lenses, the Infinity 3S camera (Teledyne DALSA, Waterloo, Ontario, Canada) and Image Pro Plus image analysis software program (Media Cybernetics, Rockville, MD, USA) and the Olympus Confocal Fluoview 1000 microscope equipped with a 60 x oil immersion lens and Fluoview software program (Olympus Corp., Tokyo, Japan). Adobe Photoshop version 23 (Adobe Inc., San Jose, CA, USA) was used to adjust photographs for brightness and contrast, and assembly and annotation of figures.
