E600 fluorescence microscope

Scratch assays as described above were performed in Lab-TekII chamber slides (Fisher Scientific, Pittsburgh, PA). Cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS), permeabilized with 0.2% Triton X-100/PBS, blocked with 5% bovine serum albumin (BSA) in PBS with 0.1% Tween-20 (PBST) at 4°C for overnight, and incubated with monoclonal anti-TG2 antibody (Neomarker/Thermoscientific, Rockford, IL) in 5% BSA/PBST at 4°C for overnight. Primary antibodies were detected with rhodamine-labeled anti-mouse IgG antibody (2 h incubation at room temperature). After washing with PBST, slides were mounted with Vectashield mounting media (Vector Laboratories, Burlingame, CA), and visualized under Nikon E-600 fluorescence microscope [29 (link)]. Nuclei were counterstained with DAPI (Sigma). TG2 on the outer surface of plasma membrane was detected as described by Akimov and Belkin [8 (link)]. Briefly, cells grown in chamber slides were incubated with anti-TG2 antibody (10 μg/ml in PBS) at 37°C for 4 h, fixed with 4% paraformaldehyde/PBS for 20 min, and blocked with 5% BSA/PBS for 2 h. Primary antibody (anti-TG2) was detected by rhodamine-conjugated goat anti-mouse IgG antibody under Nikon E-600 fluorescence microscope.

ARPE-19 cells were grown on coverslips, fixed and permeabilized with methanol at −20 °C for 10 min, and then non-specific sites were blocked by incubation with 3% BSA in Tris-buffered saline and Triton X-100 (TBST) (20 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EGTA, 0.1% Triton X-100) for 30 min. The cells were incubated with anti-acetylated-tubulin antibody to detect primary cilia (1:1000; T6793; Sigma-Aldrich; St. Louis, MO). Acetylated tubulin is a well-accepted marker for primary cilia. Cells were then incubated with FITC-conjugated donkey anti-mouse secondary antibody (1:200; 715–095-150; Jackson ImmunoResearch Inc.; West Grove, PA). DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) incorporated into the mounting media (Vector Labs; Burlingame, CA). The intracellular localization of proteins was observed with a Nikon E600 fluorescence microscope, Pan Fluor 100× objective (N.A. 0.5–1.3) or Pan Fluor 40× objective (N.A. 0.75), fit with appropriate filters and images captured with an Orca II CCD camera, model C4742–95 (Hamamatsu; Middlesex, NJ) and Metamorph image analysis and acquisition software (Molecular Devices; Sunnyvale, CA, USA). Images were exported to Photoshop (Adobe; San Jose, CA) and only linear adjustments to brightness and/or contrast were performed.

The fresh skeletal muscle and acellular white matrix were fixed in 4% paraformaldehyde for 24 hrs, rinsed with water overnight, dehydrated in graded alcohol, embedded in paraffin and sliced in 4–6 μm serial sections (Leica Microsystems, Wetzlar, Germany).
Haematoxylin and eosin and DAPI (Vector Laboratories, Burlingame, CA, USA) staining were performed after routine antigen retrieval, dewaxing and rehydrating. After conventional haematoxylin and eosin staining, sections were sealed with neutral gum and assessed by light microscopy (Olympus, Tokyo, Japan). Each section was covered with 50 μl of DAPI (0.1 μg/ml in PBS) and stored in the dark. Staining was assessed by an E600 fluorescence microscope (Nikon, Tokyo, Japan) after 5 min. Serial sections (4–6 μm) were stained with Sirius red, alcian blue, and Masson trichrome to assess the collagen and glycoprotein composition. The sections were observed by light microscopy.