Whole-mount Imaging of Bone Samples

Femurs were longitudinally cut in half, then stained, and deep imaged as described previously^{21 (link)}. The staining solution contained 10% DMSO, 0.5% IgePal630 (Sigma), and 5% donkey serum (Jackson Immuno) in PBS. Half bones were stained for 3 days at room temperature with primary antibodies. Then specimens were washed 3 times in PBS at room temperature for one day and put into staining solution containing secondary antibodies for 3 days followed by a one-day wash. Antibodies used for whole mount staining included chicken anti-GFP (Aves Labs, 1:200), anti-tdTomato (Takara, 1:200), Alexa Fluor 647-AffiniPure F(ab′)2 Fragment Donkey Anti-Chicken IgY (1:250), Alexa Fluor 488-AffiniPure F(ab′)2 Fragment Donkey Anti-Rabbit IgG (1:250), Alexa Fluor 488-AffiniPure F(ab′)2 Fragment Donkey Anti-Rabbit IgG (all from Jackson ImmunoResearch, 1:250), and 555--conjugated donkey anti-goat antibody (Invitrogen, 1:250). Images were acquired using a Leica SP8 confocal microscope. Images were rendered in 3D and analyzed using Bitplane Imaris v7.7.1.