For immunofluorescence, mice were perfused with 50 ml of PBS followed by 50 ml of 4% paraformaldehyde. The brains were harvested and fixed for 24 hrs in 4% paraformaldehyde. The samples were transferred to 30% sucrose in PBS, and frozen at OCT in −80°C until sectioning. at 40 μM sections were prepared using a Leica CM1950 cryostat. For staining of microglia, tissues were stained with goat anti-Iba-1 (Thermo), and Cy5-conjugated anti-goat secondary antibody (Jackson ImmunoResearch). Slides were imaged using a Zeiss Axio Observer fluorescence microscope with a 20X objective. The Zeiss Zen Blue software (Zeiss) was used to process the images.
For in vivo EdU labeling, mice were injected i.p. with 6 doses of EdU (100 mg/kg) over 2 days. EdU was detected by the Click-iT Plus EdU Imaging Kit (Invitrogen) according to the manufacturer’s instructions. After EdU detection, the sections were stained with anti-NeuN rabbit polyclonal ab (A60) (Millipore) and anti-rabbit Rhodamine secondary ab (Jackson). Slides were imaged using a Zeiss Axio Observer fluorescence microscope with a 10X objective. Images were processed by the Zeiss Zen Blue software (Zeiss).
For in vivo EdU labeling, mice were injected i.p. with 6 doses of EdU (100 mg/kg) over 2 days. EdU was detected by the Click-iT Plus EdU Imaging Kit (Invitrogen) according to the manufacturer’s instructions. After EdU detection, the sections were stained with anti-NeuN rabbit polyclonal ab (A60) (Millipore) and anti-rabbit Rhodamine secondary ab (Jackson). Slides were imaged using a Zeiss Axio Observer fluorescence microscope with a 10X objective. Images were processed by the Zeiss Zen Blue software (Zeiss).