Fitc conjugated

Forty-eight h post transfection, cells were fixed with methanol for 5′ and, when specified, they were further permeabilized with Triton 0.01% for 10′ a room temperature. Cells were then blocked with bovine serum albumin (BSA) 0.1% at R.T. for 30′ and incubated with the mAb570 (provided by the University of North Caroline) or the mAbCF3 (mAb 2784 Abcam) and diluted 1:500 in BSA 0.1% overnight at 4 °C. They were finally incubated with secondary anti-mouse mAb (FITC-conjugated, Sigma, diluted 1:300 or Alexa 488, Abcam, diluted 1:500) for 1 h at room temperature before DAPI staining and microscopy observation. The exposure time was adjusted using the negative control as reference (no fluorescence signal observed).

TA muscles were sectioned to 5-μm thickness. Staining with HE and FITC-conjugated wheat germ agglutinin (WGA) (1:50; Sigma) to evaluate muscle CSA, Picrosirius red to detect collagen deposition, and eMHC (1:200; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) to detect myofiber differentiation were performed as previously described (13 (link), 40 (link)). Images were captured on an ECLIPSE 90i fluorescence digital microscope (Nikon, Japan) and analyzed using NIS-Elements Br 3.0 software (Nikon).

Although there is no current consensus with respect to murine MSCs markers (versus those for human MSCs), immunophenotyping was performed by flow cytometry analysis. In brief, cells were incubated with anti-CD45.2 clone 104 (APC-eFluor780 conjugated, eBioscience), anti-CD11b, clone M1/70 (PE-Cy-conjugated, eBioscience), anti-Sca-1, clone D7 (PE-conjugated, eBioscience), anti-CD90.2 clone 53–2.1 (PE-Cy7-conjugated, BD Pharmingen^™) and anti-ASMA, clone 1A4 (FITC-conjugated, Sigma) (S2D–S2F Fig).

For morphological evaluation, endothelial cells were stained with lectin (Lectin from Bandeiraea simplicifolia, FITC conjugated, Sigma Aldrich) and AD-MSC were stained with phalloidin (Dylight^TM 554 Phalloidin, Cell Signaling Technology, Danvers, MA, USA); DAPI (Carl Roth GmbH, Karlsruhe, Germany) was used for counterstaining of nuclei. The cells were fixed with 4% paraformaldehyde (Carl Roth GmbH) for 30 min. Then the endothelial cells were permeabilized with PBS (containing Ca²⁺ and Mg²⁺) + 0.25% Triton X-100 (Carl Roth GmbH) for 30 min at room temperature. Next, 5% bovine serum albumin (Carl Roth GmbH), dissolved in distilled water, was added as a blocking solution for 30 min at 37 °C. After removing the blocking solution, the lectin (0.1 mg/ml, diluted in 0.9% saline solution) was added and incubated overnight at 4 °C. The AD-MSC were stained with phalloidin (1:200) for 30 min. Cells were washed with PBS + 0.1% Triton X-100 three times for 15 min, then DAPI (1:1000) was added for 30 min for nuclear staining. Directly after staining of the endothelial cells and AD-MSC, three images were obtained from each replicate at standardized settings using a Leica DMI6000 B microscope (Leica Microsystems, Wetzlar, Germany). Photomicrographs were analyzed using Fiji ImageJ software to determine endothelial cell counts.

For immunofluorescence analysis, HeLa cells after RBAc photodynamic treatment were fixed in paraformaldehyde (4% in PBS 0.2 M, pH 7.4) for 5 minutes. For intracellular immunofluorescence detection the cells were permeabilized with Triton (0.1% in PBS 0.2 M, pH 7.4). Permeabilized and not permeabilized HeLa cells were incubated before with polyclonal anti-CRT developed in rabbit (1∶200 in PBS/BSA0.1%; Sigma-Aldrich, St. Louis, MO, USA) for 1 h at RT and then with the anti-rabbit IgG antibody FITC-conjugated (1∶25 in PBS/BSA 0.1%; Sigma-Aldrich, St. Louis, MO, USA) for 1 h at dark and RT. Fluorescence was evaluated with a Eclipse 80i fluorescence microscope (Nikon, Tokyo, Japan).