Zen 2.3 black edition

Mouse maxillae were removed and fixed with 4% paraformaldehyde (PFA) for 24 h at 4 °C. The dissected maxilla was decalcified using 20% Morse solution (Fujifilm Wako Pure Chemical) in sterilized water for 24 h at 4 °C and subsequently incubated with 10, 20, and 30% sucrose solutions for more than 2 h at 4 °C for cryoprotection. The samples were embedded in super-cryo-embedding medium (SCEM; Section-Lab, Hiroshima, Japan). Cryosections, 16-µm-thick, were cut in accordance with Kawamoto’s film method using Cryofilm type 4D (16UF) and a tungsten carbide microtome (Section-Lab)^{44 (link)}. The sections were incubated with primary antibodies for 24 h at 24 ± 2 °C; however, in case of staining with rat anti-endomuchin antibody and goat anti-mouse-CD31antibody coupled to AF 488, the tissue was pretreated with 0.25% Triton X-100 (Sigma-Aldrich) for 10 min at 24 ± 2 °C. The sections were further incubated with secondary antibodies for 2 h at 24 ± 2 °C, mounted using 30% glycerol, covered with coverslips, and sealed with nail polish. Z stacks of confocal images were obtained at 1-µm intervals between 16-µm-thick sections. Fluorescence images were acquired using a laser-scanning confocal microscope (LSM880) equipped with a Plan-APOCHROMAT (20 × /0.8), ZEN 2.3 black edition, and ZEN 2.6 blue edition (all from Carl Zeiss, Oberkochen, Germany).

Live cell imaging was done by using Cytation 5 imaging reader at +37 °C and 5% CO₂ (BioTek^TM CYT5MPV) using the ×10 objective. Light micrographs were taken using Confocal Zeiss LSM 780 and Zeiss AxioImager.Z1 upright epifluorescence microscopes with Apotome combined with a computer-controlled Hamamatsu Orca R2 1.3 megapixel monochrome CCD camera and ZEN software (ZEN 2.3 and 2.6 Blue as well as ZEN 2.3 Black edition (Zeiss)). ×40 Plan Apochromat, 1.4 NA Oil objective was used. Post-acquisition image processing was performed using the Corel Draw software X5. Brightness and contrast were linearly adjusted using Corel Photo-Paint X8. IF and live cell imaging-based quantifications were done by using CellProfiler (3.1.8)^{65 (link)}, Qupath software (v0.1.2)^{66 (link)}, and ImageJ (1.52p) with FibrilTool plug-in^{67 (link)}.