Apochromat 20

Images described on Fig EV1B are acquired on an EVOS FL Microscope (Thermo Fisher) equipped with a GFP LED light cube. Images described on Fig EV1C are acquired on a Zeiss Ax10 Observer D1 fluorescence microscope system equipped with AxioCam and an HXP 120V lamp, with an Objective Plan‐Apochromat 20× and ZEN acquisition software.
All other fluorescent images are acquired on a Nikon Ti‐E automated epifluorescent microscope. The microscope was equipped with a DS‐Qi2 camera and a Lumencor Sola SE II 365 LED lamp. The filters sets were provided by Nikon for DAPI (DAPI‐5060C), Alexa Fluor 488 (FITC‐3540C), and Alexa Fluor 568 (mCherry C M343564, Sembrock)). The objectives used were Apo 20× Lambda, numerical aperture 0.75 (Nikon). We used a hardware‐based autofocusing system called the perfect focus system (PFS) from Nikon to automatically focus the cells in the field of view.

For awake imaging, mice were habituated to the treadmill under head-fixed conditions for 40 min per day over 2 weeks. Ca²⁺ imaging was performed using a two-photon microscope (Zeiss LSM 7 MP, Carl Zeiss, Jena, Germany) equipped with a water immersion objective lens (Apochromat 20, NA = 1.0, Carl Zeiss). Two-photon excitation at 900 nm for GCaMP6s imaging was carried out using a mode-locked Ti:sapphire laser system (Chameleon, Coherent, USA). Data were acquired using ZEN software (Zeiss Efficient Navigation, Carl Zeiss) at 4.4 Hz for imaging of S1 and 32 Hz for imaging of the cerebellar Bergmann glia.