Avidin biotinylated complex abc kit

Fresh mice cerebella were divided into right and left cerebellum. Sagittal brain slices were further sectioned at 300 μm. 1% biocytin (sigma B4261) in 2 M KCl was ionotophoretically injected into Purkinje cells by using 0.1–0.3 nA of depolarizing current for about 30 min at alternating intervals of 20 ms on and 20 ms off [29 (link)]. Brain slices were then fixed with 4% paraformaldehyde in 0.1 M PB at 4 °C for 16–18 h. Using a sliding microtome, serial frozen sections of 50 μm thickness were prepared and incubated in 0.5% H₂O₂ for 20 min to remove endogenous peroxidase. They were further blocked with blocking solution (3% Bovine serum albumin, 1% Triton X-100, 5% FBS) for 90 min at room temperature. Finally, slices were reacted with Avidin-biotinylated complex (ABC Kit, Vector) for 1 day, and developed with 3,3-diaminobenzidine. Focus was fine-tuned and all details of the cells were then depicted. Subsequently, the soma of the Purkinje cell was used as the center to quantify the maximal sagittal extension of their dendrites. Sholl analysis was applied to investigate the dendritic branches. Centered at the soma of the Purkinje cell, concentric circles with gradually increasing radii of 10 μm formed the grid. The numbers of ring-crossings of each branch (number of intersections) were counted and represented the complexity of dendritic branches [30 (link)].

Brain sections were reacted with primary antibodies of mouse anti-neural nuclei antigen (NeuN) antibody (Millipore, 1:250), mouse anti-ED1 antibody (Millipore, 1:500), or mouse anti-human-specific nuclei antigen antibody (Millipore, 1:100) at 4 °C for 12 h. Subsequently, the secondary antibody of goat anti-mouse IgG-conjugated biotin (Millipore, 1:250) was added and reacted for 1 h at room temperature. Slides were then washed with 0.1 M PBS three times (5 min each), reacted with avidin-biotinylated complex (ABC kit, Vector) for 1 h, washed three times with 0.1 M PBS (5 min each), and developed with DAB. After staining, the slides were mounted with a mounting medium (Fisher Scientific SP15-500) and examined and photographed under an optical microscope.