For immunofluorescence analysis, the dissected mouse mandibles were fixed in 4% paraformaldehyde (PFA) overnight, and then decalcified with 10% EDTA for four weeks.
Then, the tissues were dehydrated in 15% sucrose/PBS solution for two hours, then in 30% sucrose/PBS for two hours, and 30% sucrose/OCT (Sakura, Tissue-Tek, 4583) at 4°C overnight followed by embedding in OCT. Sagittal cryosections 8 μm thick were used for immunofluorescence staining following standard protocols. The primary antibodies are listed inTable S1 . Alexa Fluor 488 and Alexa Fluor 568 (1:200, Invitrogen) were used as secondary antibodies. DAPI (Invitrogen, 62248) was used for nuclear staining. All images were captured using a Keyence microscope (Carl Zeiss). For p-Igf1r, p-Irs1, p-Akt and Igf2, Alexa Fluor 488/594 Tyramide SuperBoost kits (Invitrogen, B40922/40925) were used.
Then, the tissues were dehydrated in 15% sucrose/PBS solution for two hours, then in 30% sucrose/PBS for two hours, and 30% sucrose/OCT (Sakura, Tissue-Tek, 4583) at 4°C overnight followed by embedding in OCT. Sagittal cryosections 8 μm thick were used for immunofluorescence staining following standard protocols. The primary antibodies are listed in