Af2557

For PGRN protein analysis, supernatants were mixed with equal amount of in 2× sample buffer boiled for 5 min, and resolved on a 10% SDS-PAGE gel. Proteins were transferred onto a nitrocellulose membrane and blocked over-night with membrane blocking agent (GE Healthcare) at 4°C. The blots were incubated in PBST with 1:250 anti-mouse PGRN polyclonal antibody (R&D Systems AF2557) for 1hour followed by extensive washing. After incubating with horseradish peroxidase-conjugated anti-sheep IgG secondary antibody (R&D Systems HAF016; 1:4,000) at room temperature for 1 hour, blots were visualized using enhanced chemiluminescence (GE Healthcare) and a Bio-Rad ChemiDoc MP Image System (Bio-Rad Laboratories, ON Canada). The same blot was stained with mouse monoclonal β-actin antibody (Sigma AC-40; 1:1000), as a control for total protein loading.

The frozen sections (n = 3 per group) were first permeabilized using 0.1% Triton X-100 for 10 min followed by blocking with 5% donkey serum in PBS for 30 min. The sections were then incubated with primary antibodies overnight at 4 °C, subsequently rinsed with PBS, and incubated with secondary antibodies at RT for 1 h. After rinsing with PBS for 3 times, the slides were mounted using Vectashield mounting medium with DAPI (H-1200, Vector Laboratories, Burlingame, CA, USA). The slides were subsequently imaged under immunofluorescence microscope (Axio Scope.A1, Zeiss, Oberkochen, Germany).
The primary antibodies used in this study included mouse anti-NeuN (1:400, ab104224, Abcam, Cambridge, England), mouse anti-GFAP (1:100, sc-33673, Santa Cruz Biotechnology), rat anti-CD68 (1:200, MCA1957, Bio-Rad Laboratories, Hercules, USA), sheep anti-PGRN (1:200, AF2557, R&D Systems, Minneapolis, USA), and rabbit anti-iNOS (1:200, PA1-036, Invitrogen, Carlsbad, USA). The secondary antibodies used in this study included donkey anti-mouse Alexa Fluor 488 (1:200, A-11017, Invitrogen, Carlsbad, USA), donkey anti-rat Alexa Fluor 488 (1:200, A-21208, Invitrogen, Carlsbad, USA), donkey anti-rabbit Cy3 (1:200, AP182C, Sigma-Aldrich, Darmstadt, Germany), and donkey anti-sheep Alexa Fluor 647 (1:200, A-21448, Invitrogen, Carlsbad, USA)