Tcs sp8 smd

Sections stained for c‐Fos immunostaining were photographed with a CCD camera (Model 77CE; Sony, Tokyo, Japan) attached to a microscope (Axioskop with Plan‐NEOFLUAR objectives; Carl Zeiss GmbH, Carl Zeiss, Oberkochen, Germany) using a 10× 0.63 objective. Double immunofluorescence was visualised with a confocal microscope (TCS SP8 SMD; Leica, Wetzlar, Germany) using a 40x objective. For each staining analysed, images of the areas of interest were taken at the same time under identical lighting or laser set‐ups for all animals. A person unaware of the treatments performed the quantification of either c‐Fos expression, POMC staining and co‐localisation using the ImageJ software (NIH, Bethesda, MD, USA). Positive c‐Fos staining was evaluated by setting a threshold to a mean size of the nuclear positive staining. Then the mean size was used to threshold the quantification for the rest of the images. A rectangle or circle of the size of the brain area of interest was drawn and superimposed accordingly. The POMC‐immunoreactive area was evaluated by using a set threshold and quantifying the same sampled area size for every level of the ARC. Double immunostaining was evaluated by counting exclusively POMC positive cells that contained both, c‐Fos and DAPI in the nucleus.

To stain HUH7 cells for confocal microscopy 30,000 cells/well were seeded on IBIDI μ-slides (IBIDI, Martinsried, Germany) one day before treatment with archazolid (2.5/10 nM, 24 h). After treatment, cells were washed with PBS, fixed with 3% Paraformaldehyde (PFA) for 30 min, permeabilized with 0.1% Triton-X and unspecific binding was blocked with 2% BSA. Subsequently lysosomal marker protein LAMP-1 was stained with specific antibodies (Developmental Studies Hybridoma Bank) for 2 h at 25°C and secondary antibody (AlexaFluor^®488, MolecularProbes) for 45 min at 25°C. Cholesterol was stained with 50 μg/ml filipin (Sigma Aldrich) for 2 h at 25°C, together with TO-PRO^®3 (Life Technologies) staining of nuclei (Supplementary Table S1and S2). Cells were washed and mounted with FluorSaveTM Reagent mounting medium (Beckman Coulter) and covered with a glass coverslip. Images were taken by confocal microscopy (Leica TCS SP 8 SMD, Wetzla, Germany).

To assess mitochondrial morphology by live-cell imaging, H9C2 cardiomyoblasts were plated on coverslips as described above, rinsed with PBS and incubated in phenol-red-free DMEM with 200 nM tetramethylrhodamine methyl ester (TMRM, T5428 Sigma), which accumulates in polarized mitochondria, for a minimum of 20 min. Doxycycline was removed from the medium due to its interfering effects on fluorescence signals [52 (link)]. After wash-out, coverslips were placed inverted on slides. All images were obtained with a Leica TCS SP8 SMD, mounted on a Leica DMI6000 inverted microscope enclosed in an incubator at 37 °C. Images were processed using Leica LAS-X software, and ImageJ, using the FeatureJ plugin (ImageJ 1.45s; National Institutes of Health, Bethesda, USA). Particles were analyzed for area, aspect ratio and circularity as described before [10 (link)].

Several seeding densities of C2C12 cells were tested for viability inside the 3D collagen scaffolds by dropwise addition of 100 µL complete medium containing 1 × 10⁵, 5 × 10⁵, or 1 × 10⁶ cells per UV-sterilized collagen scaffold of 12 mm. Scaffolds were put into individual wells of a 24-well plate. After cell seeding, scaffolds were incubated in a humidified incubator at 37 °C for 1 h, followed by adding 500 µL of medium and additional incubation for 48 h. For visualization of cell growth, the medium was removed, and collagen scaffolds were washed with phosphate-buffered saline (PBS). Then, 500 µL of 3 µM calcein-acetoxymethyl ester (Calcein-AM, BioLegend, San Diego, CA, USA, Cat No. 425201) and 2 µg mL⁻¹ of propidium iodide (PI, Sigma-Aldrich, Cat No. P4170) in PBS were added to the scaffolds and incubated in a 37 °C with 5% CO₂ humidified incubator for 10 min.
Directly following the incubation, live-cell imaging was performed using the Leica TCS SP8 SMD with an HCX PL APO 10×/0.40 dry objective lens and a temperature-controlled stage at 36.5 °C. Calcein-AM was excited at 496 nm using a WLL, with emission collected between 505 and 540 nm. The PI signal was sequentially acquired by excitation at 535 nm and emission collection between 605 and 645 nm.

For life cell imaging of cells, a stage top incubator (Okolab, Pozzuoli, Italy) was installed on a Leica TCS SP8 SMD. To visualize actin cells were transfected with actin-GFP (addgene 21948), actin-mCherry (addgene 54966), or nuclear actin-Chromobody® (ChromoTek GmbH, Planegg-Martinsried, Germany) using FuGENE® HD.