Dfc320

After being anesthetized, the brains were removed from the skull, and fixed in 4% paraformaldehyde overnight, and then stored at 4°C in 30% sucrose solution until they sank. The brains were freeze sectioned using a sliding microtome (Leica, Germany) into 30-μm coronal sections. For immunofluorescence staining, the slices were permeabilized in 0.3% triton for 10 min, and then blocked with 10% serum in PBS for 1 h and incubated with a primary antibody (TH, Santa Cruz, sc-374047, 1:200; Sel1l, Sangon Biotech, D161115, 1:200; Sdhc, Proteintech Group, 14575-1-AP, 1:100) overnight at 4°C. The next day, the secondary antibodies (Dylight 594-Conjugated AffiniPure Goat Anti-Mouse, Jackson ImmunoResearch, 1:400; Dylight 488-Conjugated AffiniPure Goat Anti-Rabbit IgG, Jackson ImmunoResearch, 1:400) were added to the sections. Nuclei were stained by DAPI. The slices were imaged using microscopy (Leica DFC320, Germany). The number of TH-immunoreactive positive neurons in the SNpc was counted using the previously described counting criteria[12 (link)].

The
wettability of membrane samples was assessed by measuring the
contact angle, using the sessile drop method. All types of membranes
were tested, by analyzing 6 samples for each condition. The contact
angle was measured on images acquired with an optical microscope (Leica
MZ16) equipped with a 45° tilted mirror and a digital camera
(Leica DFC 320), then connected to the software Image Pro 3D Suite.
Membrane behavior was examined in the presence of three types of fluids,
namely deionized water (DW), deionized water + 10% FBS, and DMEM.
For each type of fluid, 4 μL were deposited on the sample and
the images were obtained after 30 s to allow drop stabilization. The
acquired images were analyzed using Fiji software and the contact
angle of each type of sample in the presence of all types of fluid
was calculated. Surface free energies were evaluated using the Owens–Wendt
method^{73 (link)} adapted by Ren et al.^{74 (link)} and Can-Herrera et al.^{71 (link)} Both DW and ethylene glycol (EG) contact angles (4 μL of fluid/sample)
were considered to calculate the surface energy components: the polar/hydrophilic
component (γ_s^p) and the dispersive/hydrophobic
component (γ_s^d). The total surface free
energy (γ_s) was, therefore, calculated as

Interstitial collagen was determined in the myocardium based on Sirius red staining. Photographs of the LV wall, excluding the septum, were taken using a 40x objective (Leica DFC320). Interstitial collagen was quantified in five randomly selected areas of the subendocardium and myocardium of the LV (40x objective) and results were expressed as percentage tissue positive for Sirius red relative to myocardial area. Morphometric parameters were analyzed using a computerized morphometry system (Leica Qwin V3, Leica, Rijswijk, The Netherlands).

For in situ hybridization and in vivo protocols, images were obtained with a Leica Z6APO stereomicroscope equipped with a Leica DFC320 color camera. Immunostainings were imaged on a Zeiss LSM 710 confocal microscope. The ImageJ software was used to analyze the Z stacks and to measure regenerate area and blastema length. All statistical analyses were done on SPSS 16.0 software.

Cells were seeded in coverslips dishes at a density of l × 10⁴ cells/cm², maintained 24 h for attachment and then treated or not with BSHE (whose GR50 concentrations were those referred to the 48 h-exposure). After 48 h-incubation, cells were washed twice with PBS, stained with 4 μg/mL AO for 10 min and washed again with PBS. The coverslips were overturned on microscope slides and immediately observed under fluorescence microscopy (Axio Imager A2 Zeiss). Images were acquired using a color camera (DFC320 Leica). AO is a metachromatic dye whose luminescence wavelength is strongly dependent on its concentration. In live cells, AO is accumulated by acidic vesicles yielding strong orange or red signals; it is also provided with affinity for nucleic acids yielding less intense green fluorescence (Pierzynska-Mach et al., 2014 (link)). The vesicle red fluorescence intensity was quantified by using Fiji-ImageJ image processing software (Schindelin et al., 2012 (link)). At least 50 cells per each experimental condition were analysed, while corrected total cell fluorescence (CTCF), of both BSHE-untreated and BSHE-treated cells, was obtained as follows: CTCF = Integrated Density - (Area of selected cell x Mean fluorescence of background readings). (https://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html#measuring-cell-fluorescence-using-imagej).

Cells were seeded onto round glass coverslips of 15 mm diameter and allowed to grow up to 70% confluence. Cells were fixed with 3% paraformaldehyde (PFA) for 15 min and then incubated with a solution of 1% BSA, 0.1% Triton X-100 and 50 mM glycine in dPBS in order to perform blocking, permeabilization and quenching, for 30 min at RT. Primary antibodies diluted in dPBS + 2% BSA were added for 1 h, and secondary antibodies, for 30 min at RT. Nuclei were stained with DAPI. Glass slides were mounted with a Fluorescence Mounting Medium (Dako) and images were acquired with a Leica DM 3000 fluorescence microscope using a Leica DFC320 camera.
Spheroids in suspension were cytocentrifuged through Thermo Shandon Cytospin 3 (Thermo Scientific) at 500 RPM for 5 min. The slides were air-dried for 10 min and fixed with ice-cold acetone for 5 min. Then, the same procedure described for adherent cells was followed.

ABCB5⁺ MSCs (1 × 10⁵ and 1.5 × 10⁵) were seeded in two wells of a 24-well plate coated with Geltrex® basement membrane matrix (Thermo Fisher) and incubated in stem cell medium for 19–22 h at 37 °C, 3.1% CO₂. Tube structures were photographed using an inverted microscope (40× final magnification; DM IL LED, Leica, Wetzlar, Germany) equipped with a digital camera (DFC320, Leica) and semi-quantitatively classified into six (A–F) categories, ranging from A = tubular branches of several cells forming a defined network-like structure to F = no tubular branches visible (Fig. S2 (see Additional file 2)), with A–C being considered as successful angiogenic differentiation. For assay validation, human umbilical vein endothelial cells (Thermo Fisher) and human skin melanoma cells (SK-MEL-28, ATCC® HTB-72™, LGC Standards, Wesel, Germany) were used as positive and negative controls, respectively.