Vt100s vibrating microtome

Mice were transcardially perfused with room-temperature 0.1 M PBS, pH 7.3, followed by ice cold 4% (wt/vol) paraformaldehyde in PBS. Brains were extracted and post-fixed with 4% paraformaldehyde in PBS at 4°C. 50 µm coronal sections were cut using a Leica VT100S vibrating microtome. Goat anti-GFP (Abcam #ab6673, Waltham, MA, USA) and chicken anti-mCherry (Novus Biologicals #NBP2-25158, Littleton, CO, USA) primary antibodies were used at a 1:2000 dilution to amplify GCaMP6s and cre expression, respectively. Chicken anti-mCherry was also used to amplify virally expressed tdTomato. Donkey anti-goat conjugated to Alexa Fluor 488 (Jackson ImmunoResearch #703-545-155, West Grove, PA, USA) and donkey anti-chicken conjugated to Alexa Fluor 594 (Jackson ImmunoResearch #703-585-155) secondary antibodies were used at a 1:1500 dilution. The Brain BLAQ protocol (Kupferschmidt et al., 2015 (link)) was used for immunohistochemistry following electrophysiology.

Cerebellum slice cultures were performed according to a previous publication.
²³ (link) Irradiated or nonirradiated cerebella were embedded in 3% tissue culture grade agarose, and 300 μm sagittal slices were cut using a VT100S vibrating microtome (Leica). Slices were then transferred to a 0.4 μm Nuclepore membrane (Millicell) at the interface between air and culture medium (NB‐B27 medium) in a 6‐well culture plate and incubated at 37°C in 5% CO₂. In certain experiments, cerebellum slices derived from nonirradiated mice were treated with 200 U/mL recombinant mouse IFN‐γ (Peprotech) for 24 h during culture, and then tissue lysates were prepared for Western blotting and qPCR. Cerebellum slices derived from irradiated mice 12 h post irradiation were treated with an IFN‐γ neutralizing antibody (clone R4‐6A2, BioXcell) or isotype control rat IgG1 for 24 h at 10 μg/mL, and then tissue lysates and frozen sections were prepared for Western blotting and immunohistochemistry, respectively.