Mice were deeply anaesthetised with pentobarbital ip and then perfused transcardially with ice-cold PBS, pH 7.4, followed by 4% paraformaldehyde (PFA, Sigma-Aldrich, France). Brains were extracted and post-fixed in 4% PFA overnight at 4 °C. The next day, brains were washed 3 times 2 h in PBS. For delipidation, brains were first incubated in 50% CUBIC-L reagent (T3740, Tokyo Chemical Industry, Japan) diluted in water for 24 h at 37 °C and then in 100% CUBIC-L reagent for 5 days. CUBIC-L solution was refreshed on days 2 and 4. Brains were then washed 3 times 2 h in PBS at RT. The following step consisted in refractive index matching and started with 50% CUBIC-R (T3741, Tokyo Chemical Industry, Japan) diluted in water for 24 h at RT followed by 100% CUBIC-R for 24 h at RT. CUBIC cleared brains were acquired on a Ultramicroscope II light sheet microscope (LaVision Biotec, Germany) using a sCMOS camera (Andor Neo) and a 2x/0.5 objective lens.
Leon S., Simon V., Lee T.H., Steuernagel L., Clark S., Biglari N., Lesté-Lasserre T., Dupuy N., Cannich A., Bellocchio L., Zizzari P., Allard C., Gonzales D., Le Feuvre Y., Lhuillier E., Brochard A., Nicolas J.C., Teillon J., Nikolski M., Marsicano G., Fioramonti X., Brüning J.C., Cota D, & Quarta C. (2024). Single cell tracing of Pomc neurons reveals recruitment of ‘Ghost’ subtypes with atypical identity in a mouse model of obesity. Nature Communications, 15, 3443.
+ Open protocol