Sp8 inverted confocal laser scanning microscope

Double Alcian blue/Alizarin red S staining on fixed zebrafish was performed as previously described^{42 (link)}. The sample size (n) is 5 embryos per each group. The zebrafish palate and lower jaw were dissected and mounted in 4% methyl cellulose prior to imaging. Tartrate- resistant acid phosphatase (TRAP) staining for osteoclast activity was performed (n = 5 wild-type and 5 rspo3^−/−) as adapted from previous study^{43 (link)}. Imaging was performed using Nikon Eclipse 80i microscope (Melville, NY, USA) and NIS-Elements Br imaging software version 4.40 (2015). Measurements were taken in ImageJ. In vivo Alizarin red S staining of 9 dpf zebrafish was performed as previously described^{44 (link)}. Alizarin red S and sox10:kaede fluorescence was imaged using a Leica SP8 inverted confocal laser scanning microscope. Maximum intensity projections of z-stacks were generated using ImageJ version 2.0.

For sarcomere staining in vitro, myotubes were differentiated on Nunc Lab-Tek Chamber Slides (Thermo Scientific) and fixed in 4% paraformaldehyde at day 6 of differentiation. Staining was performed by blocking slides with 0.5% Triton-X 100 in PBS, and incubating α-actinin antibody (R&D, MAB9830; 1:400) overnight at 4 °C. The next day, fibers were incubated with secondary antibody (1:500) and DAPI (1:1000) at room temperature for one hour. Fibers were imaged using a Leica SP8 inverted confocal laser scanning microscope and sarcomere length was assessed using the Find Peak plugin for ImageJ on the staining histogram.

MEFs were cultured overnight on glass coverslips in six-well plates. After transfection and/or virus infection, cells were washed with PBS, fixed with paraformaldehyde (ThermoFisher Scientific) for 15 min, and washed three times with PBS. Cells were then permeabilized with 0.5% Triton-X in PBS for 5 min, followed by three PBS washes. Cells were blocked in 3% bovine serum albumin for 1 h. MEFs transfected with Myc-AIP plasmids were stained with mouse anti-Myc antibody (1:1000 in 3% bovine serum albumin) overnight. On the following day, cells were washed three times with PBS before incubation with Alexa Fluor 594–conjugated goat anti-mouse secondary antibody (1:1000, ThermoFisher Scientific). Coverslips were washed six times with PBS and then mounted onto slides using ProLong Diamond Antifade Mountant with DAPI staining (ThermoFisher Scientific). Images were obtained with a Leica SP8 Inverted Laser Scanning Confocal Microscope using a 63× oil objective. Images were analyzed using Fiji and JaCOP plugin for colocalization analysis. Nuclear translocation was quantified by analyzing the ratio of GFP in the nucleus to GFP fluorescence in the entire cell using Fiji.