Alexa fluor 488 conjugated goat anti rabbit secondary antibody

T-cell lines were cytospun onto slides at 800 rpm for 5 min. The cells were then fixed in 3.7% paraformaldehyde (PFA) for 15 min at RT, washed with PBS, permeabilized on ice for 5 min with 0.5% Triton X-100, and blocked for 1 h in PBS with 0.5% gelatin and 0.25% bovine serum albumin at room temperature. Slides were next incubated with anti-iNOS and anti-p-H2AX antibodies at 1/200 in PBS for 2 h, washed three times in PBS-0.2% gelatin for 10 min each time, and incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody and Alexa Fluor 596-conjugated goat anti-mouse secondary antibody (Molecular Probes, Invitrogen) in PBS-0.2% gelatin for 1 h at room temperature. Cells were washed three times in PBS-0.2% gelatin for 10 min each time and mounted using DABCO mounting medium (2.5% DABCO from Sigma, 200 mM Tris–HCl pH 8.6 and 90% glycerol). Fluorescent images were captured using a Nikon Eclipse Tis epifluorescence microscope and the NIS elements software (Nikon). The images were collected by using the objectives 20X and 40X, and the number of DNA Double-Strand Breaks (DSB) foci and the quantification of fluorescence intensity was performed using the object count module in NIS elements.

Immunofluorescence for γ-H2AX, a DNA damage and repair marker, was also evaluated. Briefly, cells were exposed to 0 or 5 Gy of γ-ray. At the indicated times after irradiation, cells were washed with cold PBS and fixed with 4% formalin for 10 min at room temperature. After washing three times with PBS, cells were incubated with a rabbit anti-human γ-H2AX polyclonal antibody (1:500 dilution, Abcam) overnight at 4°C. After washing three times with PBS, cells were incubated with an Alexa Fluor® 488 conjugated goat anti-rabbit secondary antibody (1:250 dilution; Molecular Probes Inc., USA) at room temperature for 2 hrs in the dark. The immunofluorescence for γ-H2AX in cells was examined on an Olympus fluorescent microscope, and the number of γ-H2AX foci per cell was counted under the microscope with 400-fold magnification. The mean number of γ-H2AX foci from more than 50 cells was calculated for statistical analysis.

Seven weeks after spinal cord injury, rats were deeply anesthetized using 10% chloral hydrate, perfused transcardially with ice-cold heparinized (10 U/mL) saline, and then injected with 4% PFA in PBS. The spinal cord was dissected, and fixed in 4% PFA solution in 0.1 M PBS for 48 h at 4°C. The 8-mm-long spinal cord tissue centered on the lesion was frozen and sliced in 20 μm thick. In brief, the sections were rehydrated in PBS for 5 min followed by incubation with 1% bovine serum albumin and 0.01% triton-X100 for 1 h at room temperature. Then the sections were exposed to appropriate primary monoclonal antibodies (1:300 Tuj-1, Abcam; 1:300 GFAP Abcam; 1:500 MBP; 1:200 Synaptophosin) in a humidified chamber at 4 °C overnight. After washing three times with PBS, the slides were then incubated with an Alexa Fluor 594-conjugated goat anti-mouse secondary antibody (1:800, Molecular Probes) or Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:400, Molecular Probes) at 4 °C in the dark for 2 h. Finally, the sections were mounted onto glass slides for microscopic analysis after staining with 4',6-Diamidino-2-phenylindole (DAPI) and following extensive rinsing with PBS. The cellular and morphological structures of the spinal cord tissue were assessed by H&E staining.