Dmi8 inverted fluorescence microscope

Dissociated cultures were fixed in 4% paraformaldehyde (PFA Sigma Aldrich) in phosphate‐buffered saline (PBS) for 15 min at room temperature (RT). Next, cultures were washed three times in 0.5% PBS, and cells were permeabilized with 0.5% Triton X‐100 in PBS for 10 minutes at RT. To block non‐specific binding, cells were incubated in 5% normal goat serum (Sigma) in PBS for 1 h. Primary antibodies including polyclonal rabbit Tuj 1 (T2200, anti‐βIII tubulin, Sigma, 1:1000), chicken anti‐GFAP (Ab4674, Abcam 1:1000), rabbit anti‐NeuN (Ab104225, Abcam, 1:1000) and rabbit anti‐S100 (Ab34686, Abcam, 1:1000) were applied for 1 h at RT. Then, cultures were washed with PBS 3 times and incubated with Alexa Fluor 488‐conjugated goat anti‐rabbit, Alexa Fluor 568‐conjugated goat anti‐rabbit and Alexa Fluor 647‐conjugated goat anti‐chicken secondary antibodies (Thermo Fisher Scientific; 1:500 in 1% BSA/PBS) for 1 h in the dark. Finally, the cells were washed 3 times with PBS and mounted with Vectashield mounting medium containing DAPI (Vector Labs).
Cell cultures were imaged using a DMi8 inverted fluorescence microscope (Leica microsystems) coupled to a monochrome digital camera (Hammamatsu C4742‐374 95) and a Leica SPE confocal microscope with 63x oil immersion objective and Leica LAS AF software. Image stacks taken were analysed and edited using Fiji software.

The hASCs of induced (chondro) and non-induced (control) experimental conditions of TCP or MatriXpec™ plating were initially fixed with 4% PFA and then washed with PBS. Cells were stained with 0.1% Safranin O solution (Sigma-Aldrich, St. Louis, MO, United States) diluted in deionized water for 30 min at room temperature. After the removal of the Safranin O solution, samples were washed. The plates were analyzed at ×20 magnification in a DMi8 inverted fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

Confocal imaging was performed using DMi8 inverted fluorescence microscope (Leica Microsystems) equipped with a 40 × 1.3 NA oil-immersion objective. DAPI, BSA-Alexa488, and Alexa647 were excited with 405 nm, 488 nm, and 638 nm laser diodes, respectively. The imaging was performed in sequential mode (each channel separately), to avoid fluorescence bleed-through between the channels. The emitted light was collected by a hybrid detector after: 410–450 nm (for DAPI), 510–550 nm (for BSA-Alexa488), and 650–750 nm (for Alexa647–conjugated antibodies) bandpass.
Bright-field imaging of fibrinogen/cresyl violet staining was performed using BX53 upright transmitted light microscope (Olympus) equipped with a 60 × 1.4 NA oil-immersion objective.
All images were exported to ImageJ (NIH, version 1.48u4) for further analysis. For comparisons between WT and Apom^-/- mice, the images were collected using the same microscope settings, and Z-projected using maximal signal intensity projection from an equally thick section (=10 µm). The WT vs. Apom^-/- mice imaging data on the figures are presented in the fluorescence signal intensity scale.

The induced (chondro) and non-induced (control) samples were initially fixed with 4% PFA, followed by three washes with 1X PBS. Then, cells were stained with 0.1% Safranin O solution (Safranin O diluted in deionized water) for 30 min (Sigma-Aldrich, St. Louis, MO, USA). After the removal of the Safranin O solution, plates were washed three times with 1X PBS to remove any excess dye. The plates were analyzed at 20× magnification under a DMi8 inverted fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

All fluorescence microscopy experiments were performed using a DMi8 inverted fluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with a 100×, 1.47 numerical aperture (NA) Plan-Apochromat oil immersion lens, a Flash 4.0 v3 sCMOS camera (Hamamatsu, Shizuoka, Japan), an LED3 fluorescence illumination system, 488 nm and 561 nm lasers, a W-View Gemini image splitting optical device (Hamamatsu), compatible filter sets for fluorescence and DIC imaging and the LAS X v3.7.6.25997 software (Leica). Image acquisition parameters (illumination intensity, exposure time and camera binning) were consistently applied within each trial of an experiment to allow comparison between strains.
Images were processed following acquisition by importing them into Fiji/ImageJ2 v2.9.0/1.53t. Linear adjustments were made to minimum and maximum intensity levels for each 16-bit image within an experiment prior to conversion into 8-bit format for figure preparation.

The mandible samples were decalcified in a fast decalcified solution (Biotech, China) for 24 h at room temperature, dehydrated in graded ethanol and then embedded in wax. The tissue was cut into 5 μm sections using a microtome (Leica) and stained with HE, Masson’s trichrome and TRAP with commercial staining kits (Servicebio) to assess new bone formation and osteoclast function. After being blocked with bovine serum albumin (Servicebio), the sections were incubated with primary antibodies against Arg-1 (1:500, GB11285, Servicebio) and Runx2 (1:500, GB11264, Servicebio) as well as CD86 (1:500, 26903-1-AP, Proteintech) and Osx (1:500, ab209484, Abcam) at 4 °C overnight. Then, the sections were incubated with appropriate secondary antibodies (Servicebio) before mounting with DAPI. Samples were imaged using a DMI8 inverted fluorescence microscope (Leica Microsystems, Germany) and an LSM980 laser scanning confocal microscope with quantitative analysis by ImageJ.