Fitc donkey anti mouse igg h l

Tissue sections were washed three times using ice-cold 0.3% Tween 20 (Sigma-Aldrich, P1379) in PBS and blocked with 10% normal donkey serum (Jackson ImmunoResearch, West grove, PA, USA) in PBS containing 0.3% Triton X-100 (Sigma-Aldrich, X100) for 1 hr at RT. The blocked sections were incubated overnight with diluted primary antibodies including mouse anti-HuN (1:300; Abcam, Cambridge, MA, USA), mouse anti-CD68 (1:300; Abcam), goat anti-GFAP (1:500, Abcam), chicken anti-MBP (1:400, Abcam), rabbit anti-Tuj1 (1:2000, Abcam), rabbit anti-IL-1β (1:500, Abcam), rabbit anti-NT-3 (1:200, Abcam), and rabbit anti-GDNF (1:100, Abcam) in 10% NDS at 4 °C. After incubations, tissues were washed three times with ice-cold 0.3% Tween 20 and treated with species-specific secondary antibodies, including FITC-donkey anti-mouse IgG (H + L) (1:150, Jackson ImmunoResearch), CyTM3-donkey anti-rabbit IgG (H + L) (1:600, Jackson ImmunoResearch), DyLightTM405-donkey anti-chicken IgY++ (H + L) (1:600, Jackson ImmunoResearch), and CyTM5-donkey anti-goat IgG (H + L) (1:200, Jackson ImmunoResearch) for 1 hr at RT. The slide-mounted tissue was coverslipped using mounting solution with or without DAPI (Vector Laboratories, Inc., Burlingame, CA, USA). Fluorescence imaging was observed and documented using a scanning confocal laser microscope (LSM700, Carl Zeiss, Oberkochen, Germany).