Rabbit anti mouse caspase 3

Total proteins from HUVEC and irradiated intestine were prepared using RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) plus 1× Protease Inhibitor Cocktail (Sigma-Aldrich), 1× Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich) and 1× Phosphatase Inhibitor Cocktail 3 (Sigma-Aldrich). Hot-denatured proteins were used for the immunoblotting experiment. Primary antibodies included rabbit anti-mouse total Akt (Cell Signaling Technology, MA, USA), rabbit anti-mouse phosphorylated Akt Ser473(Cell Signaling Technology), rabbit anti-mouse Bcl-xL(Cell Signaling Technology), anti-mouse Bax(Cell Signaling Technology), rabbit anti-mouse caspase 3(Cell Signaling Technology), rabbit anti-mouse cleaved caspase 3(Cell Signaling Technology), rabbit anti-mouse SDF1(Cell Signaling Technology) and rabbit anti-mouse GAPDH (Cell Signaling Technology). Gray density of each sample was analyzed by using Image-Pro Plus Software (Media Cybernetics, Rockville, MD, USA).

Whole lung tissue isolated from the mice were first homogenized in isotonic buffer with CHAPS detergent (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM CHAPS) with protease inhibitors (100 μM 3,4-dichloroisocoumarin (DCI), 10 μM trans-epoxysuccinyl-L-leucylamido- (4-guanidino) butane (E-64), 2 mM o-phenanthroline monohydrate (all from Sigma) and then sonicated briefly. The protein concentration of the total lung homogenates was quantified using the BCA standard protein assay kit (Thermo Fisher). Lung homogenate samples (50μgs) were separated by SDS-PAGE and transferred to PVDF membranes as described previously [37 (link)]. The membranes were blocked overnight in 5% nonfat dry milk/PBST for an hour at room temperature. The membranes were then incubated with rabbit anti-mouse caspase-3 (1:1000; Cell Signaling) overnight at 4°C followed by anti-rabbit HRP IgG (1:2000) for 1 hr at room temperature. The membranes were washed with PBST (3x for 10 min) between primary and secondary antibody incubation. The reactive bands were visualized using the chemiluminescence method (SuperSignal West Pico, Pierce). The same membranes were reprobed with GAPDH (1:500; Cell Signaling) as loading controls. Densitometric analysis was performed using ImageJ to compare protein levels and the data analyzed by unpaired two-tailed student T test.

The animals were anesthetized and perfused intracardially with ice-cold 0.1M PBS (pH 7.4) followed by 4% paraformaldehyde. The cerebrums were post-fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and cut into 5 μm coronal sections. The paraffin-embedded sections were heated at 95°C in 0.01 M sodium citrate buffer solution (pH 6.0) for 30 min to repair the antigen. The sections were blocked in 5% bovine serum albumin in PBS containing 0.2% Triton at 25°C for 2 h, and then incubated with mouse anti-mouse NeuN (ab104224, Abcam, 1:1000 dilution) and rabbit anti-mouse caspase-3 (Cell Signaling Technology, 9661S; 1:1000) as primary antibodies at 4°C overnight. After washing thrice with PBS, the sections were incubated with biotinylated goat anti-mouse 488 (A11001, Invitrogen, Carlsbad, CA, 1:500 dilution) and goat anti-rabbit 546 (A11010, Invitrogen, Carlsbad, CA, 1:500 dilution) secondary antibodies at room temperature for 1 h. The stained sections were imaged with a fluorescence microscope (Model BX-61, Olympus, Center Valley, PA, USA). Sections without antibody staining were used as negative controls.