Fluorescent mounting medium

To observe morphological changes, U251 cells were plated in 12-well plates and treated with indicated concentrations of ARP. Images were obtained with an inverted phase contrast microscope attached to a video camera and captured by National Institutes of Health (NIH) imaging software ImageJ. For confocal microscopy, U251 cells were plated on sterile cover slips in 12-well plates and treated with 25 or 50 µM ARP. After 24 h, cells were washed with phosphate-buffered saline (PBS), fixed with 3.7% formaldehyde, and washed three times with PBS. Blocking was with 1% bovine serum albumin (BSA) in PBS, and cells were treated with Hoechst dying solution (1:1000) for nuclear staining. Coverslips were washed with PBS and mounted on glass slides using fluorescent mounting medium (DakoCytomation, Carpentaria, CA, USA). For cytoskeleton staining, FITC-phalloidin (Molecular Probes, 1:250) was added in 1% BSA and incubated for 1 h in the dark. Coverslips were washed three times with PBS. Alexa 488-conjugated secondary antibody (1:100) in 1% BSA was added and incubated for 1 h with shaking at room temperature. Coverslips were washed three times with PBS and mounted onto slides using fluorescent mounting medium (DakoCytomation, Carpentaria, CA). Intensity changes in DAPI and the cytoskeleton were imaged with an Olympus LX70 FV300 (Olympus, Tokyo, Japan).

Cells were seeded at a cell density of 30,000/cm² in a 48 well plate on cover slips. The plates were fixed at different time points with 4% paraformaldehyde (Roth, Karlsruhe, Germany) for 10 min at room temperature and then washed twice with PBS. For immunofluorescent stainings, the cells were permeabilized with 0.2% triton-X in PBS. After washing twice with PBS, the cells were blocked in 1% BSA, 2% horse and goat serum, and 0.05% Tween in PBS for 1 h. Primary antibodies were diluted in blocking solution and incubated over night at 4°C (see Supplementary Table 1 for list of antibodies). The next day, the cells were washed twice with PBS-T and treated with secondary antibodies also diluted in blocking solution. After 2 h, cells were washed twice with PBS-T, then incubated for 10 min with DAPI (Thermo Fisher, Waltham, MA, United States; 1:1,000 in PBS) and washed with PBS. The cover slips were fixed on object slides with fluorescent mounting medium (Agilent Technologies, Santa Clara, CA, United States). The staining was analyzed using the Leica DMI6000B microscope (Leica Microsystems, Wetzlar, Germany) with a 40x air objective. Images were processed with the ImageJ software (Schindelin et al., 2012 (link), 2015 (link)) and the brightness and contrast was set to the same values in all pictures from one staining. Cells were counted using the Cell Counter in the ImageJ software.

Immunofluorescence staining was performed after 7 days of drug treatment to investigate tissue integrity and presence of cell-specific markers. Membranes were excised from biochips and cells were fixed with ROTIHistofix 4% (Carl Roth) for 10 min at RT. Permeabilization and blocking was performed by adding PBS (Lonza) containing 0.1% saponin (Carl Roth) and 3% normal donkey serum (Abcam, Cambridge, UK) for 30 min at RT. The membrane was subsequently divided with scissors to independently stain the vascular and hepatic cell layers with the primary antibody solution. Primary antibodies ASGPR1 (BD Biosciences, Heidelberg, Germany), CYP3A4 (Sigma-Aldrich), α-GST (BIOZOL, Eching, Germany), CD32b (BIOZOL) and CD206 (Abcam) were incubated at 4 °C overnight. Membranes were washed with PBS/0.1% saponin and incubated with secondary antibodies DAPI (Thermo Fisher Scientific), donkey-anti-mouse-AF647 (Thermo Fisher Scientific), donkey-anti-rabbit-Cy3 (Jackson ImmunoResearch, Cambridgeshire, UK) and donkey-anti-goat-AF488 (Thermo Fisher Scientific) for 1 h at RT. Stained membranes were washed twice with PBS/0.1% saponin, once with PBS and lastly with AQUA AD iniectabilia (B. Braun, Melsungen, Germany). Samples were embedded in fluorescent mounting medium (Agilent Technologies, Waldbronn, Germany).

To study the engraftment of human UC-MSCs in mouse renal tissues, HNA was used. Briefly, 8 μm thick sections from OCT-included kidney specimens were fixed 10 min in cold acetone at 4 °C. Then, sections were incubated with 1% BSA blocking solution in PBS and then incubated with anti-human Nuclei-Fluor 488-conjugated antibody (1:50; #MAB1281A4 clone 235-1; Merck Millipore) at 4 °C overnight. In addition, a mouse anti-human mitochondria (hMT) Cy3-conjugated antibody (1:50; #MAB1273C3 clone 113-1; Merck Millipore) was used. Nuclei were counterstained with DAPI and the renal structure was marked with Alexa Fluor 633-Conjugated WGA (1:100; #W21404; Thermo Fisher, Life Technologies) for 10 min Cover slips were mounted using fluorescent mounting medium (Dako, Agilent Technologies, Santa Clara, CA, USA). Twelve sections for each animal (n = 5) were analyzed at confocal microscope (Zeiss, Original magnification ×630), and HNA-positive cells were counted. Data were expressed as the number of HNA-positive cells per 1 × 10⁵ renal cells.

For immunofluorescence imaging, cells grown on glass coverslips were fixed in 4% paraformaldehyde, washed three times with PBS, permeabilised with 0.5% Triton X-100 in PBS for 10 min and washed again three times with PBS. Coverslips were then blocked with 3% BSA in PBS for 1 h, incubated with the mouse monoclonal anti-EGFR antibody (Santa Cruz USA, #sc-101; dilution: 1/100), washed three times with PBS, stained with rabbit anti-Mouse IgG (H + L) (Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermofisher USA, A11059, USA; dilution; 1/2000)), washed again three times with PBS, counterstained with Hoechst (1/5000) and finally mounted with a fluorescent mounting medium (Agilent, USA) under a glass coverslip. Images were acquired with a Zeiss LSM 880 confocal microscope (Zeiss, Germany).

Spinal cultures were treated for 1 h or 15 min at 37°C with fibulin-2 protein at 25 or 50 nM. Then, cultures were treated for 30 min at 37°C with baclofen (10^–3 M to 10^–9 M, Sigma B5399), or with GABA (10^–4 M, Sigma A5835); or with competitive antagonists CGP55485 (0.5 or 50 μM, Tocris 55845) or saclofen (100 μM or 1 mM, Sigma S166) co-applied with baclofen. Next, cells were fixed 20 min at 37°C [4% paraformaldehyde, 4% sacharose in phosphate buffer (PB) 0.1M, pH 7.4], washed [0.1M PB saline (PBS), 10 min at RT] and mounted on slides (Fluorescent Mounting Medium Dako Cytomation, Agilent, Santa Clara, CA, United States).