M165fc dissecting microscope

All animals were euthanized by immersion in a 168 mg/L tricaine-S solution (MP Biomedicals) prepared in tank water prior to fixation. For staining on fixed tissues, larvae (8 dpf, 6 mm SL and 10 mm SL) were fixed in 4% PFA and stained with alizarin red and alcian blue or alcian alone as described^{57 (link)} with 60 mM MgCl₂. Fixed adults were stained as described^{58 (link)} with modifications^{57 (link)} for acid free stain. Live bone staining by calcein green and alizarin red was conducted as described previously^{59 (link)} with modifications.^{60 (link)} Fish were stained for 24 hours in calcein green, returned to tanks for 14 days, and then chased with alizarin red for 24 hours. Fish were then returned to tanks for 24 hours before fixation overnight in 10% normal buffered formalin at 4° C and stored in 100% ethanol in the dark at 4°C until trypsin digestion as previously described.^{60 (link)} Preparations were phenotyped on either a Leica M165FC dissecting microscope or a Zeiss Axioskop 2 FS Plus compound microscope. The dissecting microscope was equipped with a Planapo 1.6X M-series objective, a Prior L200 fluorescent light source and a Leica DC7000T camera controlled by LAS X software. The compound microscope was equipped with a Qimaging Micropublisher 6 camera controlled by Ocular software.

Transgenic lines were imaged using a Leica DM2500 compound microscope equipped with a Leica DFC500 camera, a Leica M165FC dissecting microscope equipped with a DFC340 FX camera, or a Zeiss 700 confocal microscope. Transgenic fish were fixed for 4 hours at 4°C in either 4% paraformaldehyde in 1X PBS or 10% neutral buffered formalin. For Alizarin red fluorescent counterstaining of GFP lines, 0.01% Alizarin red was added to the fixative. Tooth number was counted on the DM2500 with TX2 filter to visualize Alizarin-stained teeth. Tooth germs with GFP+ epithelia were counted on photographs of GFP fluorescence.