A11304

Brains were sectioned coronally at 10 μm at approximately bregma −2.00 mm. Serial 10 μm sections immediately rostral and caudal to the section mounted on the Visium Spatial Gene Expression slide (10X Genomics) were collected for immunohistochemistry and stored in cryoprotectant at −20 °C. Primary and secondary antibodies were diluted in 3% normal goat serum (LAMPIRE Biological Laboratories #7332500) with 0.2% Triton X-100 (Sigma CAS #9036-19-5). The tissue was blocked in 10% normal goat serum with 0.2% Triton X-100. Sections were incubated overnight at 4 °C with rabbit anti-P2ry12 (Anaspec #AS-55043A, 1:400), rat anti-GFAP (Invitrogen #13-0300, 1:400), followed by PBS wash and incubation with goat anti-rat AF568 (Invitrogen #A11077, 1:400) and goat anti-rabbit AF488 (Invitrogen #A11304, 1:200) for 2 h at room temperature. The sections were then washed, mounted on slides, and allowed to dry overnight. The slides mounted with dried tissue were then incubated in X-34 (Sigma #SML1954) 10μg/mL solution for 10 min at room temperature before being washed in PBS and differentiated in 80% ethanol for 1 min. The slides were coverslipped with ProLong Gold Antifade Mountant (ThermoFisher #P10144) and imaged on a Zeiss Axio Scan Z1 digital slide scanner at 20× magnification.