Ab15690

The brains of the mice were removed and fixed in PFA at 4°C overnight. Subsequently, they were transferred to a sucrose solution of gradually increasing concentration for 48–72 h. The brain samples were then sliced into 20 μm thick coronal sections using a CryoStar™ NX50 Cryostat (Thermo Fisher Scientific, Waltham, USA). For immunochemistry, the brain sections were incubated overnight with primary antibodies including anti-NeuN, anti-GFAP, anti-IBA1, anti-CD31 and anti-TuJ1 (Abcam, ab104224, ab7260, ab15690 and ab28364, and R&D, MAB1195) at 4°C overnight. The sections were rinsed with phosphate buffered saline (PBS) twice, followed by incubation with the corresponding secondary antibodies (Thermo Fisher Scientific, A11008, A11001) at room temperature for 2 h and then rinsed with PBS. Finally, the slices were covered with DAPI (ZLI-9557, ZSGB-BIO, China) and images were captured using a Zeiss fluorescence microscope (LSM 800, Germany).

Rats were deeply anesthetized with 1% pentobarbital sodium by i.p. injection and quickly perfused with 0.9% saline followed by 4% formaldehyde. The spinal cords of the L4-L6 section were extracted and fixed in 4% formaldehyde for 12 h at 4 °C. Samples were immersed in 30% sucrose in 0.1 M PBS at 4 °C for 3-4 days. Then, the 25-um-thick sections of spinal samples were prepared using a cryostat (CM1950; Leica, Germany). After blocking in 3% bovine serum albumin (BSA) solution containing 1% Triton X-100 for 1.5 h at room temperature, sections were washed in PBS and then stained with primary antibodies (anti-Iba-1 (1:500, Abcam, ab15690), anti-TREM2 (1:1000, R&D System, MAB17291)) for 12-14 h at 4 °C followed by corresponding secondary antibodies (1:1000, Invitrogen). Images were captured using a confocal microscope (Nikon, Japan). Immunofluorescence density was analyzed by using the software ImageJ.