Alexa fluor 488 or 594

For immunofluorescence staining of the mice brain tissues, the isolated tissues were fixed with 4% paraformaldehyde for 24 h, then stored in 70% ethanol at 4°C. The fixed tissues were embedded in paraffin. Paraffin sections (5 μm) were blocked with PBS containing 3% bovine serum albumin (Santa Cruz Biotechnology) for 30 min at room temperature and incubated with primary antibodies followed by incubation with either anti-rabbit or mouse IgG conjugated to Alexa Fluor 488 or 594 (Thermo Fisher Scientific). For immunofluorescence staining of cultured cells, the cells were fixed with 10% formalin in PBS for 10 min, blocked with PBS containing 3% bovine serum albumin for 30 min at room temperature, then incubated with the primary antibodies followed by incubation with either anti-rabbit or mouse IgG conjugated with Alexa Fluor 488 or 594. The samples were examined by a fluorescence microscope (model BX41; Olympus) and camera (model DP80; Olympus) at room temperature.

Mice were deeply anesthetized with a ketamine-xylazine mixture (100 and 10 mg/kg body weight, respectively, intraperitoneally) and perfused with 4% paraformaldehyde in 0.1 M phosphate-buffer (PB). The brains were removed from the skull and post-fixed in the same fixative overnight. Subsequently, the brains were cryoprotected in 20% sucrose/PB overnight, frozen, and cut to a thickness of 50 μm on a cryostat. The sections were rinsed with phosphate-buffered saline containing 0.1% Triton X-100 (PBST) and incubated with primary antibodies overnight at room temperature. The following antibodies were used: goat anti-GFP (1:250, #600-101-215, Rockland), mouse anti-CaMKII (1:200, ab22609, Abcam), rabbit anti-GFAP (1:1000, G9269, Sigma-Aldrich), mouse anti-GFAP (1:1000, G3893, Sigma-Aldrich), and rabbit anti-DsRed (1:1000, #632496, Clontech). For fluorescence microscopy, the sections were treated with a mixture of species-specific secondary antibodies conjugated to AlexaFluor-488 or -594 (1:500, Thermo Fisher Scientific) for 2 hat room temperature. Fluorescence images were obtained using a fluorescence microscope (BZ-X800, Keyence).

Formalin-fixed and paraffin-embedded arterial tissue sections were used after antigen retrieval by heating in microwave oven in Tris/EDTA buffer pH9 (Dako). buffer. For single labeling, CARD9 rabbit polyclonal antibody (Abcam, Cambridge, UK) was used at 1:500 dilution, incubated for 1 h and revealed using ABC-peroxidase technique (Vector Laboratories, Burlingame, CA, USA). For double labeling, the sections were first incubated with mouse monoclonal anti-CD68 antibodies diluted at 1:50 (to detect macrophages) or anti-α smooth muscle actin antibodies diluted at 1:100 (to detect smooth muscle cells), both from Dako-Agilent (Trappes, France). Sections were then incubated with species-specific secondary antibodies (1:500 dilution, 45 min at room temperature) conjugated to AlexaFluor 488 or 594 (Fischer Scientific) and mounted on microscope slides using the Prolong Antifade Diamond kit (Thermofisher). Image acquisition was performed on a laser scanning confocal microscope (Leica TCS SP8, Leica Microsystems).

Specimens were pre-treated for 5 minutes in 0.01 M citrate buffer, pH = 6.0 at 90 °C, autofluorescence was quenched by 15 min incubation in 0.1 M Glycine-tris-buffered saline (TBS) at room temperature (RT), then unspecific binding was blocked with 10% Normal Goat Serum/0.5% Triton-TBS solution for 30 min at RT. Primary antibodies TBS-dilutions were applied overnight at +4 °C. As primary antibodies, we used rabbit anti-BDNF (Santa Cruz, sc-546, dilution 1:100) and mouse anti-GFAP (Sigma, G3893, dilution 1:500). Species-matching secondary antibodies (AlexaFluor 488 or 594, Thermo, dilution 1:500) were applied for 3 hours at RT. Nuclei were counterstained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Sigma). Samples were evaluated using the Zeiss Axio Scope.A1 fluorescent microscope (Zeiss, Oberkochen, Germany).

Muscle sections were hydrated in distilled water, washed with PBS, and fixed in 4% paraformaldehyde in PBS at room temperature for 10 min. The samples were then washed twice with PBS, permeabilized with 0.3% Triton X-100 (Sigma Aldrich) in PBS at room temperature for 10 min and incubated in PBS containing 3% BSA (Sigma Aldrich), 2% donkey serum (DS; Sigma Aldrich) and 5% FBS at room temperature for 1 h. Next, all samples were incubated with primary antibodies: anti-CD45 (Abcam ab25386) or anti-CD68 (Abcam, ab53444) diluted in blocking buffer in PBS (1:100) at 4 °C overnight, followed by incubation with appropriate secondary antibodies conjugated with Alexa Fluor 488 or 594 (Thermo Fisher Scientific), diluted 1:200 in PBS with 3% BSA, 5% FBS, and 2% DS at room temperature for 1 h. The nuclei were visualized with Hoechst (Sigma-Aldrich) diluted 1:1000 in PBS for 10 min at room temperature. The samples were mounted with fluorescent mounting medium (Dako Cytomation, Glostrup, Denmark). The number of CD45+ or CD68+ cells was counted 14 days after cell transplantation. A total of 20 fields of view of 3 muscles from each experimental variant were analyzed with ImageJ software.

Cells (30,000–80,000) were seeded over glass coverslips placed into 24-well plates prior to treatment. At desired end-points, cells were fixed by 30 min incubation in 4% (w/v) paraformaldehyde (Sigma-Aldrich) in PBS. Cells were sequentially permeabilized with 0.1% (w/v) SDS in PBS (USB Corp.) and subjected to antigen blocking with 10% (v/v) FBS in PBS (both for 20 min). Primary antibodies employed were Bax, p21^Waf1/Cip1, p16^INK4a and phospho(Ser10)-Histone H3 (Table 2). Secondary antibodies were Goat anti-Mouse or anti-Rabbit IgG (H + L) Secondary Antibodies, conjugated either with Alexa Fluor^® 488 or 594 (ThermoFisher Scientific: #A-11001, #A-11005, #A-11008 and #A-11012). DNA counterstaining was performed upon 10 min incubation with 5 μg/mL solution of DAPI (Molecular Probes). Photographs were taken with either an Axio Observer.Z1 (Carl Zeiss AG) or a DM6000B (Leica Microsystems) epifluorescence microscope. Images were analyzed with Image J software (freely available from the National Institutes of Health at the address https://imagej.nih.gov/ij/).