4 6 diamidino 2 phenylindole (dapi)

Spleen and tumor tissues were harvested, cut into small pieces, embedded in OCT freezing medium (Sakura Finetek, Torrance, CA), and stored in − 80 °C until further use. Tissue sections (8 μm thick) were fixed in chilled acetone for 5 min, air dried, washed with cold PBS, and blocked with 10% normal horse serum (Jackson ImmunoResearch, West Grove, PA) at room temperature (RT) for 30 min. Then washed with PBS and incubated with indicated fluorochrome-conjugated primary Abs (1:200 dilution for FITC anti-mouse F4/80, 1:400 dilution for rabbit monoclonal Ab to iNOS (Abcam) and 1:100 dilution for Alexa Fluor 488 anti-mouse CD206, and Alexa Fluor 647 anti-mouse F4/80) on ice for 45 min. Followed by three-time washing with cold PBS, and incubated with secondary antibodies (1:1000 dilution for donkey Dylight 549 anti-rabbit antibody) for 30 min; then washed three-times with PBS, fixed with 1% paraformaldehyde, and mounted in aqueous mounting medium containing DAPI (Electron Microscopy Sciences, Hatfield, PA). Immunofluorescence images were captured using the Leica DMI 6000 fluorescent microscope (Leica Microsystems, Germany), and data were analyzed using Leica AF6000 software.

RT112 and UM-UC-3 cells were seeded on chamber slides (#154461; Nalgene Nunc International, Rochester, NY) at 50% confluency. The following day, cells were washed with PBS and fixed in 4% paraformaldehyde at room temperature for 15 min. Cells were permeabilized and blocked with 1% BSA and 0.5% Triton X-100 in PBS at room temperature for 30 min. Chamber slides were incubated with anti-PAI-1 antibody (H-135; Santa Cruz Biotechnology, Santa Cruz, CA, 1:50 dilution) diluted in 1% BSA and 0.5% Triton X-100 in PBS at room temperature for 1 h. Cells were washed with PBS and subsequently treated with anti-rabbit DyLight 633 antibody (Catalog #: 35562; Thermo Fisher Scientific, Waltham, MA, 1:280 dilution) at room temperature for 1 h in the dark. Chamber slides were washed and mounted with mounting media containing DAPI (#17984-24; Electron Microscopy Sciences, Hatfield, PA). IF images were captured using a Leica TCS SP5 confocal fluorescence microscope at 400X magnification (Leica Microsystems, Bannockburn, IL).

Cells were washed with ice-cold PBS (pH 7.4), followed by 4% PFA for 15 min. After washing with PBS, cells were incubated with 3% BSA in PBS for 1 h and then incubated with primary antibody against ZO-1 (1:200; Invitrogen, Carlsbad, CA, USA) and Claudin-5 (1:200; Invitrogen, Carlsbad, CA, USA) at 4 °C overnight. After washing with PBS, they were incubated with anti-rabbit secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 568 (1:500; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Finally, nuclei were counterstained with DAPI (Electron Microscopy Sciences, Hatfield, PA, USA).

Cross sections of TA muscles obtained as described above were blocked (1 h) in blocking solution and processed as described previously [58 (link)] for detection of atrogin, Cx43 or Cx45. Antigen sites recognized by each antibody were detected with appropriate dilutions of Cy2- or Cy3-conjugated goat anti-rabbit IgGs (1:1000). Then, samples were washed 4 times with PBS 1X and once with distilled water, after which they were mounted with Fluoromount-G™ and nuclei were stained with DAPI (Electron Microscopy Science, Hatfield, PA, USA) and mounted on glass slides. The images were acquired in a Nikon Eclipse Ti microscope, and fluorescence intensity quantification was carried out using ImageJ. 10 images for each sample of at least 3 animals were analyzed per condition.