Immunohistochemistry and immunofluorescence

All procedures were performed as our previously described studies (15). Briefly, mice were perfused with saline, and brains were fixed with 4% paraformaldehyde (w/v) for 1 week. Fixed mouse brains were cryoprotected in 30% sucrose. Coronal sections were cut, and sections were stained with GFAP (1:1000; Sigma-Aldrich, catalog no. G3893), Iba1 (1:500; WAKO, catalog no. NCNP24), CD68 (1:500; Abcam, catalog no. ab53444), TREM2 (1:50; R&D Systems, catalog no. AF1729), and Aβ (1:500; Covance, catalog no. SIG-39320).

The quantification of immunohistochemistry (IHC) was analyzed by the software of Image-Pro Plus (Media Cybernetics Inc.). For quantification of Calhm2 ISH levels, the spot number of Calhm2 staining in each cell was counted in the indicated brain area. For Iba1 or GFAP IHC levels, we have counted all the intact cells from same pixel area in the indicated brain area.