Mab360

Mice was perfused with 4% PFA and the cervical spinal cord was dissected. Next, the tissues were fixed in 4% PFA overnight at 4°C, followed by processing in 20% sucrose, embedded in OCT, and then sectioned into 20 μm, and subjected to tissue staining after cryosection. A blocking solution was prepared with 0.1% Tritox-100, 3% goat serum, 0.1% BSA plus PBS solution, and blocked at room temperature for 1 h (Xiang et al., 2020 (link)). After blocking, sections were incubated overnight at RT with the specific primary antibodies (anti-CXCR3, 1:5,000, 26756-1-AP, Proteintech; anti-NeuN, 1:200, Millipore, MAB377; anti-GFAP, 1:500, Millipore, MAB360; anti-CD11b, 1:200, Abcam, ab8878). Then, they were washed 3 times in PBS, and incubated with the appropriate secondary antibodies (Alex Fluor™ 647 goat anti-rabbit IgG (H + L), 1:1,000, Thermo Fisher Scientific, #1851447; Alex Fluor™ 568 goat anti-mouse IgG (H + L), 1:300, Thermo Fisher Scientific, 1862187) for 1 h at RT. After incubated with 5 μg DAPI, tissue sections were washed, mounted and then imaged after drying. All imaging were performed on a Zeiss Axio Observer 7 Fluorescence Microscope. The immune signal intensity was quantified in a semi-automatic manner using Image-Pro Plus software and 4–5 images were counted for each mouse as described previously (Zhao et al., 2013 (link); Xiang et al., 2020 (link)).

After being anesthetized with euthatal (from Merck) (60 mg/kg), the Nrg1-reporting mice (2 months old) were transcardially perfused with PBS (2 ml/g of body weight), followed by 4% PFA in PBS. Brains were harvested, incubated in 4% PFA overnight, and dehydrated at 4 °C in two steps with 20% and 30% sucrose in PBS. Brains were frozen in OCT (catalog #14-373-65; Fisher) and sectioned into 40 µm slices on a cryostat microtome (Bosch Microm HM550) at − 20 °C. Brain slices were permeabilized with 0.3% TritonX 100 and 5% BSA in PBS and incubated with primary antibodies at 4 °C overnight. The brain slices were not treated with Triton- × 100 when staining with anti-GAD67 antibodies. After washing with PBS for three times, samples were incubated with Alexa Fluor-488 or -647 secondary antibodies (1:1000, ThermoFisher Scientific) for 1 h at room temperature. Samples were mounted with Vectashield mounting medium (Vector) and images were taken by Leica TCS SP8 confocal microscope. The following primary antibodies were used: rabbit anti-NeuN (1:500, Abcam, ab177487), rabbit anti-TH (1:250, Millipore, MAB152), mouse anti-NRGN (1:200, R&D, MAB7947), mouse anti-PV (1:500, Sigma, P3088), mouse anti-GFAP (1:250, Millipore, MAB360); rabbit anti-S100β (1:200, Abcam, Ab52642) and mouse anti-GABA (1:1000, Invitrogen, PA5-32241).

The antibodies used are anti-IL-6 (1:2500, Abcam, ab7737); anti-IL-1β (1:5000, R&D, AF-401-NA); anti-Wnt5a (1:2500, Abcam, ab72583); anti-CD11b (1:2500, Abcam, ab133357); anti-GFAP (1:10000, Millipore, MAB360); anti-GAPDH (1:10000, Abcam, ab181602); anti-α-tubulin (1:10000, Proteintech, 66031-1-lg); Goat Anti-Rabbit IgG H&L (HRP) (1:30000, Abcam, ab97051) and Goat Anti-Mouse IgG H&L (HRP) (1:30000, Abcam, ab97023).

Four weeks post-injection, animals were transcardially perfused with 1× phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA). Brains were extracted and subsequently fixed in 4% PFA overnight at 4 °C. Brains were then immersed in 30% sucrose (prepared in 1× PBS), at 4 °C, until equilibrated in sucrose mixture. Brains were embedded in a 1:2 OCT (Tissue Tek, Torrance, CA) and 30% sucrose mixture, and cryo-sectioned at 40 μm (Cryostar NX70, ThermoScientific, Waltham, MA). Sections were permeabilized in 0.5% TritonX-100 for 1 h, blocked in 5% goat serum (10% normal goat serum, 50062Z, Life Technologies) for 1 h, and then incubated in primary antibody (anti-NEUN, 1:1000, EMD Millipore MAB377; anti-GFAP, 1:500, EMD Millipore MAB360; anti-OLIG2, 1:200, Abcam ab109186; anti-IBA1, 1:1000, Wako Chemicals NC9288364; anti-GFP, 1:800, Invitrogen A11122) overnight at 4 °C. Sections were washed three times in 1× PBS and incubated in secondary antibody (anti-mouse, Invitrogen A32744; or anti-rabbit, Invitrogen A32740) for 1 h at room temperature. Sections were washed three times in 1× PBS and mounted with Vectashield containing DAPI (Vector Laboratories, Burlingame, CA).