The free-floating Etoposide ic50 sections were preincubated in 2% bovine serum albumin (BSA) diluted in PBS containing 0.3% Triton X-100 (PBS-Triton X-100 0.3%) for 30 min. Double immunofluorescence of GFAP and NF-L, was carried

out after a two day incubation at 4 °C with rabbit polyclonal anti-GFAP and mouse monoclonal anti-NF-L (clone NR-4), diluted 1:3000 and 1:2000, respectively, in PBS- Triton X-100 0.3%. For Neu-N immunofluorescence, the sections were incubated two overnights at 4 °C with mouse polyclonal anti-NeuN diluted 1:1000 in PBS-Triton X-100 0.3%. The negative controls were performed omitting the primary antibodies. After washing several times in PBS, tissue sections were incubated with anti-rabbit Alexa 488 and anti-mouse Alexa 568, both diluted 1:500 in PBS-Triton X-100 0.3% for 1 h at room temperature (for GFAP and NF-L immunofluorescence). Other tissue sections were incubated with anti-mouse Alexa 488, diluted 1:500 in PBS-Triton X-100 0.3% for 1 h at room temperature (for Neu-N immunofluorescence). Afterwards, the sections were washed several times in PBS, transferred to gelatinized slides, mounted with Fluor Save™ (Merck Rio de Janeiro, RJ), covered

with coverslips and sealed with nail polish. The images were obtained with an Olympus IX-81 confocal FV-1000 microscope and analyzed Ku-0059436 mouse with an Olympus Fluoview software. Tissues were dissociated with PBS/Collagenase/DNase, washed once with PBS then suspended in PBS/collagenase containing 10 μg/ml propidium iodide (PI). The integrity of plasma membrane was assessed by determining the ability of cells to exclude PI. The cells were incubated at room temperature in the dark for 30 min, washed with PBS and centrifuged at 3000 rpm for 5 min at 4 °C to remove Tyrosine-protein kinase BLK the free PI. Afterwards, the cell was permeabilized with 0.2% PBS Triton X-100 in for 10 min at room temperature and blocked for 15 min with BSA 5%. After blocking, cells were incubated in blocking solution containing the monoclonal antibodies anti-NeuN (clone A60) diluted 1:100 or anti-GFAP diluted 1:100, for 2 h. The cells were washed twice with PBS and incubated for 1 h in blocking solution containing

fluorescein isothiocyanate (FITC)-anti-rabbit IgG diluted 1:200 or Alexa 488-anti-mouse IgG diluted 1:200. The levels of PI incorporation, levels of positive NeuN cells and positive GFAP cells were determined by flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA). FITC or Alexa Fluor 488 and PI dyes were excited at 488 nm using an air-cooled argon laser. Negative controls (samples with the secondary antibody) were included for setting up the machine voltages. Controls stained with a single dye (Alexa fluor 488 or FITC and propidium iodide) were used to set compensation. The emission of fluorochromes was recorded through specific band-pass fluorescence filters: green (FL-1; 530 nm/30) and red (FL-3; 670 nm long pass).