Tsc sp8 confocal laser scanning microscope

immunofluorescent staining was performed as described previously by us^{46 (link),47 (link)}. Briefly, 2 × 10³ cells were plated into chamber slides (Millipore), and then were incubated with primary antibody against LC3 or p-AKT overnight at 4 °C. After washing with 1× PBS for three times, cells were incubated with secondary antibodies conjugated with Alexa Fluor 594 for 1 h. The slides were washed three times with 1× PBS, counterstained with DAPI, mounted and stored at 4 °C under dark conditions. Autophagy was quantified by quantification of LC3 puncta per cell using Leica TSC SP8 confocal laser scanning microscope. The average number of LC3 puncta per cell was counted with at least 100 cells for each cell line. The images were taken under confocal laser scanning microscope (LC3) or Leica DMI4000 fluorescenc microscope (p-AKT).

Cryostat sections (7 μm thick) of homograft tumor tissues containing EGFP-P29 and MitoB LT Red-labeled A11 cells were fixed with 4% paraformaldehyde and stained with rabbit polyclonal anti-GFP antibody (Proteintech, Sankt Leon-Rot, Germany) followed by Alexa Fluor 488 (AF488)-conjugated anti-rabbit IgG. In this case, to clearly visualize EGFP-P29 cells, immunostaining with anti-GFP antibody was employed. Cryostat sections of tumor tissues containing P29 and MitoB LT Red-labeled A11 cells were fixed with 4% paraformaldehyde for 10 min and blocked with 1% BSA in DPBS. The sections were treated with an M. O. M. Immunodetection Kit (Vector Laboratories, Burlingame, CA, USA) and then incubated with mouse monoclonal anti-α-SMA (clone 1A4) (Dako, Jena, Germany) antibody followed by AF488-conjugated goat anti-mouse IgG. The sections were counterstained with DAPI and observed under a Leica TSC SP8 confocal laser scanning microscope. WA-mFib cells were also immunostained with the anti-α-SMA antibody followed by AF594-conjugated goat anti-mouse IgG.

Using a Leica TSC SP8 confocal laser scanning microscope and the tissue’s PFA-induced autofluorescence, images were generated at a speed of 400 Hz and 2048 pixels × 2048 pixels with a fivefold object lens or 1024 pixels × 1024 pixels with a 10-fold object lens. Following the previously published protocol, the cochlea was scanned and the images were exported and further processed using ImageJ software (Wrzeszcz et al., 2013 (link)). The area of Rosenthal’s canal was traced and the SGNs were automatically counted in the traced area using the Image-based Tool for Counting Nuclei (ITCN) plug-in (Center for Bio-Image Informatics¹). SGN count was performed on five subsequent images of cochlear cross-sections. The number of SGNs divided by the measured cross-sectional area of Rosenthal’s canal gives the SGN density (cells/10,000 μm²). The mean SGN density of the total cochlear length, including all cross-sections of Rosenthal’s canal, was analyzed. The mean SGN density of the (lower and upper) basal turn was also analyzed, but separately.

TEM and STEM observations were taken on a FEI Titan Cubed Themis G2300 transmission electron microscope with an acceleration voltage of 300 Kv. Carbon coated copper grids were dipped in the chloroform solutions to deposit the samples onto the films. UV-visible spectra were recorded on an HP8453 UV-visible spectrophotometer. X-ray powder diffraction (XRD) patterns were obtained by using a Philips pw1830 X-ray diffractometer. X-ray photoelectron spectroscopy (XPS) were obtained by using K-Alpha+. Dynamic light scattering (DLS) measurement was performed by NanoBrook ZetaPlus. Confocal images were taken using a Leica TSC SP8 confocal laser scanning microscope (Germany) with a 63× oil-immersion objective.

Glioblastoma cells were seeded onto sterile coverslips in 24-well plates (2.5 × 10⁴ cells per well) and incubated for 24 h at 37 °C. After 3-h inhibitor treatment cells were fixed with 4 % paraformaldehyde for 15 min and then permeabilized with 0.25 % Triton X-100 for 10 min at room temperature. Coverslips were blocked with 1 % bovine serum albumin (BSA) for 30 min, then incubated with Alexa Fluor 594-conjugated phalloidin (Invitrogen) for 20 min to visualize actin filaments. Cells were air dried and mounted with ProLong Gold antifade mountant with DAPI stain (Invitrogen). Lamellipodia were analyzed under Leica TSC SP8 confocal laser scanning microscope and membrane ruffles were observed by phase contrast microscopy. In each case, about 200 cells were photographed and representative cells were shown. Independent experiments were carried out in triplicate.