Gel mount aqueous mounting medium

Cells were prepared for immunofluorescence (IFA) as previously described.12 Following subsequent washing with phosphate buffered saline (PBS) ×3 (5 minutes each), cells were incubated with the appropriate secondary antibodies (Alexa Flour 568 donkey anti‐goat immunoglobin G; Molecular Probes, Eugene, OR, USA) and diluted in Dako Real Antibody Reagent (Dako, Glostrup, Denmark). After washing, cells were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma Aldrich, St. Louis, MO, USA) for five minutes, washed with PBS ×3 (5 minutes each), mounted in Gel Mount Aqueous Mounting Medium (Sigma Aldrich) and examined by fluorescent microscope (Olympus, Tokyo, Japan).

Mouse hindlimbs were pinned to cork, immersed in ice-cold relaxing buffer, relaxed for 24 h at 4°C, and then fixed in 4% PFA in relaxing buffer for 24 h at 4°C. TA and soleus muscles were then excised, cryoprotected in 30% sucrose in PBS for 4 h, embedded in Tissue-Tek OCT Compound (Sakura Finetek USA, Torrance, CA), and frozen on a metal block chilled in liquid N₂. Muscles were divided into 12-μm-thick cryosections, mounted on slides, and stored at −20°C until use. Sections were washed in PBST, permeabilized for 15 min in PBS plus 0.3% Triton X-100, and blocked for 2 h in 4% BSA plus 1% goat serum in PBST. Tissues were labeled with primary antibodies diluted in blocking buffer overnight at 4°C, washed three times in PBST, and then labeled with a fluorophore-conjugated secondary antibody mixture in blocking buffer for 2 h at room temperature. The secondary antibody mixture was supplemented with rhodamine–phalloidin (1:100; Life Technologies) to stain F-actin. Tissues were then washed three times in PBST, preserved in Gel Mount aqueous mounting medium (Sigma-Aldrich), and coverslipped.

Following DMSO or 30 µM 15d-PGJ₂ treatment at 37°C, the CGTH W-2 cells were washed with PBS, then fixed for 5 min with 10% formalin in PBS and permeabilized for 10 min with 0.1% Triton X-100 in PBS at room temperature. Following the PBS washes (3 times for 5 min each), nonspecific binding was blocked with a 30 min incubation at room temperature with 5% non-fat milk in PBS. The cells were incubated overnight at 4°C with monoclonal mouse antibodies against p120-ctn (dilution, 1:100; cat. no., 610133) or β-ctn (dilution, 1:100; cat. no., 610153; BD Biosciences, Franklin Lakes, NJ, USA), or vinculin (dilution, 1:100; cat. no., V4505; Sigma-Aldrich; Merck Millipore) washed with PBS and incubated for 1 h at 37°C with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (dilution, 1:50; cat. no., 715–095-151; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). For F-actin labeling, the cells were incubated with FITC-phalloidin (dilution, 1:100; cat. no., P5282; Sigma-Aldrich; Merck Millipore). Finally, the cells were washed with PBS, mounted with Gel Mount™ Aqueous mounting medium (Sigma-Aldrich; Merck Millipore) and detected using a fluorescent microscope (DM2500; Leica Microsystems GmbH, Wetzlar, Germany) and images were captured using a Nikon D1X digital camera (Nikon Corporation, Tokyo, Japan).