Vectashield dapi 4 6 diamidino 2 phenylindole 2hcl

Mouse brain was fixed in 4% formaldehyde for 24 h, and then incubated in 30% sucrose until tissues are sink. Fixed brain was flash frozen using pre-cooled isopentane (− 78 °C), sectioned at 30 μm using Microm HM525 Cryostat (Thermo) and picked up on Superfrost Plus slides (VWR, 48311-703). Sections were blocked with 5% normal goat serum and washed in PBS with 1% bovine serum albumin (BSA) and incubated with rabbit- anti-mouse Iba-1 primary antibody (FUJIFILM Wako Pure Chemical Corporation 019-19741, 1:500) or rabbit- anti-mouse Iba-1 primary antibody Alexa Fluor^® 594 conjugate (Cell Signaling, 48934, 1:50) overnight. Sections were washed with phosphate buffer with 1% Tween 20 (PBS-T), and then incubated in goat anti-rabbit IgG (H + L) secondary antibody (Vector laboratory CY-1300, 1:250) at room temperature for 1 h when unconjugated antibody was used. Afterword, sections were washed three times with PBS-T followed by mounting on coverslip using Vectashield DAPI (4′6-diamidino-2-phenylindole 2HCl, Vector Labs, Burlingame, U.S.) mounting media to detect nuclei.

Mouse brain was fixed in 4% formaldehyde for 24 hours, and then incubated in 30% sucrose until tissues are sink. Fixed brain was flash frozen using pre-cooled isopentane (−78 °C), sectioned at 30 μm using Microm HM525 Cryostat (Thermo) and picked up on Superfrost Plus slides (VWR, 48311–703). Sections were blocked with 5% normal goat serum and washed in PBS with 1% bovine serum albumin (BSA) and incubated with rabbit- anti-mouse Iba-1 primary antibody (FUJIFILM Wako Pure Chemical Corporation 019–19741, 1:500) or rabbit- anti-mouse Iba-1 primary antibody Alexa Fluor^® 594 conjugate (Cell Signaling, 48934, 1:50) overnight. Sections were washed with phosphate buffer with 1% Tween 20 (PBS-T), and then incubated in goat anti-rabbit IgG (H+L) secondary antibody (Vector laboratory CY-1300, 1:250) at room temperature for 1 hour when unconjugated antibody was used. Afterword, sections were washed three times with PBS-T followed by mounting on coverslip using Vectashield DAPI (4′6-diamidino-2-phenylindole 2HCl, Vector Labs, Burlingame, U.S.) mounting media to detect nuclei.