Cy3 conjugated anti rabbit igg

Immunocytochemical procedures were performed as described previously [17 (link)]. Briefly, cells were collected by pipetting with PBS and plated on coverslips coated with poly-L-lysine (Sigma-Aldrich) and fixed. After incubation in the digestion buffer with or without protease-free Chase ABC (1 U/mL, Seikagaku Corporation) at 37°C for 2 h, cells were incubated in blocking solution containing 2% bovine serum albumin, 2% horse serum, and 2% goat serum, and in primary antibody (unsulfated CS stub antibody, 1B5, mouse IgG1, Seikagaku Corporation) at 4°C overnight. The 1B5 antibody recognizes a disaccharide neoepitope generated at the non-reducing terminal of CS chains that were pre-digested with Chase ABC [20 (link)]. After three washes with Tris-buffered saline, the cells were then treated with Alexa Fluor 488-conjugated anti-mouse IgG1 (Molecular Probes). The nuclei of cells were counterstained with 4’,6’-diamidino-2-phenylindole (DAPI, Sigma).
For CD133 antibody, after incubation in blocking solution containing 10% donkey serum at room temperature for 1h, cells were incubated in primary antibody (CD133, rabbit, Abcam) at 4°C overnight. After three washes, the cells were then treated with Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch) and DAPI (Sigma).
Quantification of immunocytochemical staining was described in S1 File.

Cells were grown on coverslips in 12 or 24-well plates. Following treatment, cells were fixed using 4% formaldehyde and permeabilized using 0.2% Triton X-100. After blocking with 4% BSA, cells were incubated with anti-Tom 20 antibody (Cell signaling) and Phalloidin-Alexa488 (Invitrogen), followed by the Cy3-conjugated anti-rabbit IgG (Jackson Immuno Research or Invitrogen), and observed using a confocal microscope (Zeiss, LSM700 or LSM780). To quantify perinuclear mitochondria, the ratio of Cy3 fluorescence intensity of the region surrounding nucleus region (within 5 μm) to the total intracellular Cy3 fluorescence intensity was calculated using ZEN microscopy software.

A series of coronal sections were selected to perform immunochemical staining of BrdU, nestin, DCX, NeuN, and laminin. The primary antibodies were applied including sheep polyclonal anti-BrdU (1 : 500, Abcam, USA), mouse monoclonal anti-BrdU (1 : 1000, Sigma-Aldrich, USA), mouse monoclonal anti-nestin (1 : 1000, Chemicon, USA), mouse monoclonal anti-neuronal nuclei (NeuN, 1 : 1000, Chemicon, USA), or rabbit polyclonal anti-laminin (1 : 200, Sigma-Aldrich, USA). After blocking with goat serum and incubation with primary antibodies overnight at 4°C, the slices were incubated with fluorescent-labeled secondary antibodies Alexa Fluor 488-conjugated anti-sheep IgG (1 : 200), Cy3-conjugated anti-mouse IgG (1 : 200), or Cy3-conjugated anti-rabbit IgG (1 : 200; all three antibodies from Jackson Immunoresearch Laboratories, USA) for 1 h at room temperature. For BrdU immunofluorescence, brain sections were pretreated for 30 min at 37°C with 2 N HCl and then rinsed for 10 min in 0.1 M boric acid (pH = 8.5) at room temperature followed by incubation with blocking solution. Phosphate-buffered saline instead of primary antibody was applied in negative control. The images were taken from four different fields at least (Olympus BX51; Olympus).