Fluorsavetm

For immunocytochemistry, cells cultured on 10-mm coverslips were washed once with pre-warmed PBS and subsequently fixed with 4 % PFA for 10 min at 37 °C. After fixation, the motoneurons were permeabilized with blocking buffer containing 0.1–0.3 % Triton X-100 and 10 % donkey serum in PBS for 30 min at room temperature. Primary antibodies were diluted in blocking buffer and incubated over night at 4 °C. Cells were then washed 5 times for 5 min each with washing buffer (0.1 % Triton X-100, 0.2 % Tween-20 in PBS) and incubated with secondary antibodies diluted in freshly prepared blocking buffer for 1 h at room temperature. Subsequently, the motoneurons were washed again twice for 5 min, followed by 10 min of DAPI (0.4 µg/ml) staining. After three additional washes, the coverslips were washed again once with ddH₂O and mounted on glass slides using FluorSaveTM (Millipore).

As for DAB staining, mice were transcardially perfused with 4% PFA and postfixed for an additional 24 h. Following immersed into 4% agarose, brains were sectioned into 30 μm thick sagittal sections using the VT1000S vibratome (Leica Microsystems, Wetzlar, Germany). Tissue sections were incubated with 0.1% Triton/PBS and 1 M glycine, followed by blocking with 1% BSA in PBS for 45 min. The sections were then incubated with the primary antibody AT8 (1:100, Invitrogen, CA, USA) overnight. This was followed by washing with PBS and a second incubation for 2 h at room temperature with a second primary antibody against NeuN (1:400, Millipore, Arklow, Ireland), GFAP (1:400, Sigma-Aldrich, Arklow, Ireland), Iba-1 (1:400, Wako, Fuggerstrasse, Germany) or Zinc Transporter 3 (ZnT3; 1:200, Synaptic Systems GmbH, Goettingen, Germany). After washing in PBS, tissue was incubated with fluorescent secondary antibodies [AlexaFluor-488 or AlexaFluor-568 (BioSciences, Dublin, Ireland)] followed by a short incubation with DAPI. FluorSave^TM (Millipore, Arklow, Ireland) was used to cover the tissue, and confocal images were taken with a TCR 6500 microscope (Leica Microsystems, Wetzlar, Germany) equipped with four laser lines (405, 488, 561, and 653 nm) using a 40× immersion oil objective (NA 1.3; Leica Microsystems).

Indirect immunofluorescence of hADSCs obtained from mechanically activated adipose tissue was performed as previously reported by us [39 (link)]. Briefly, 3.5 × 10³ cells/cm² were seeded onto glass slides, grown until 85% confluence, and then fixed with 4% paraformaldehyde. Saturation and permeabilization was performed with 4% BSA and 0.3% Triton X-100. Then, cells were incubated for 18 h at 4 °C with primary antibodies directed against Vimentin (Polyclonal, Santa Cruz, CA, USA), FABP4 (Polyclonal, Cell Signalling, Danvers, MA, USA), and Osteocalcin (Polyclonal, Abcam, Hongkong, China). Cells were washed with PBS and then probed for 45 min with secondary antibodies Alexa Fluor 488 or 543 (Invitrogen, Carlsbad, CA, USA). Nuclei were counterstained with DAPI (2 μg/mL in PBS, Roche, Basel, Switzerland), and glasses were mounted with FluorSaveTM (Millipore, Burlington, MA, USA). Immunofluorescence was analyzed by using a Leica SP2 confocal microscope with He/Kr and Ar lasers (Heidelberg, Germany), which was also used for image acquisition. In negative controls, primary antibodies were replaced with equivalent concentrations of unrelated IgG of the same subclass [39 (link)].