Anti calbindin

Frozen coronal sections of olfactory bulb were initially permeabilized with PBS and 0.3% Triton X-100 (Sigma-Aldrich) for 30 min, and incubated with blocking solution (0.3% Triton X-100 and 3% BSA (Sigma-Aldrich) in PBS) for 2 h at room temperature to reduce nonspecific background. The sections were then incubated with primary antibodies and diluted in PBS containing 0.3% Triton and 1% BSA overnight at 4℃. After this, the sections were washed with PBS for 3 times, and incubated with secondary antibodies for 2 h at room temperature. Finally, the sections were mounted using Prolong Gold Antifade Mountant (Thermo Fisher Scientific). The primary antibodies used were: anti-calbindin (1:30, rabbit, Cat No. ab108404, Abcam), and anti-hACE2 (1:150, goat, Cat No. AF933, R&D Systems). Fluorescent secondary antibodies conjugated to either Alexa 594 (1:200, Invitrogen) were used to detect the primary antibodies. DAPI (Invitrogen) was used to stain the nuclei.

Mice were deeply anesthetized and perfused with ice-cold 4% paraformaldehyde, brains were dissected out and equilibrated with 30% sucrose overnight. Cerebellum was sectioned using a Leica cryostat (CM3050). For immunofluorescence analysis, serial sections were incubated with blocking solution (5% normal serum in 0.3% Triton X-100 in PBS) and then with the following antibodies overnight at 4 °C: anti-calbindin (1:500, Abcam, Cat #ab82812, lot. GR110079, and Santa Cruz Biotechnology, Dallas, TX, Cat #sc-7691, lot. G2512), anti-mGlu5 receptor (1:200, Abcam, Cat #AB76316, lot. GR45647-18) and anti-mGlu1α receptor (1:100, BD Biosciences, Cat #556389, lot. 3172791). Sections were also incubated with anti-mGlu5 receptor^{42 (link)} (1 μg/ml) and anti-type-8 carbonic anhydrase^{43 (link)} (1 μg/ml). After washing, sections were incubated with secondary antibodies conjugated with Alexa Fluor 488 (1:200, Molecular Probes, Eugene, OR, Cat #S32354) and Cy3 (1:200, Jackson ImmunoResearch Labs, West Grove, PA, Cat #715-167-003) for 2 hours at room temperature and rinsed in PBS. Finally, sections were mounted with anti-fading agent (Vector Laboratories, Burlingame, CA) and examined with ZEISS 780 confocal laser scanning microscope. We used a 488 nm argon laser to excite alexa488 and 543 HeNe laser to excite Cy3.