Anti akt and pakt

The rats were sacrificed by decapitation at 2 and 8 weeks P.I. (n = 3 per time point per group). The whole brain cortex and a 10-mm lumbar SC segment from each rat were harvested for Western blot analysis. Proteins were separated by 12% SDS-PAGE electrophoresis and were blotted onto a polyvinylidene difluoride membrane. The membrane was subsequently incubated with rabbit polyclonal anti-GFAP (1:200; Thermo Fisher Scientific, Waltham, MA, USA), anti-AKT (1:500; R&D Systems, Minneapolis, MN, USA), anti-Tie2 (1:500; R&D Systems, Minneapolis, MN, USA), anti-AQP4 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-AKT and pAKT (1:500; R&D Systems, Minneapolis, MN, USA), anti-Tie2 (1:2000; R&D Systems, Minneapolis, MN, USA), anti-αvβ3 and α5β1 integrins (1:1000; Neuromics, MN, USA), anti-caspase 3 (1:500; Cayman Chemical, Ann Arbor, MI, USA), and mouse anti-MBP (1:1000; Abcam, Cambridge, MA, USA) primary antibodies for 12 h at room temperature. To normalize protein bands to a gel loading control, the membranes were re-probed with rabbit anti-β-actin antibody (1:5,000; Abcam, MA, USA). The membranes were then incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:5,000; Santa Cruz, CA, USA) and the blots were detected using enhanced chemiluminescence reagent. The incubation step with the primary antibody was omitted for the negative control.

After fixing cultured cells with 4% paraformaldehyde for 30 min, the cells were washed with phosphate-buffered saline (PBS) and later incubated with primary antibodies against polyclonal rabbit anti-TSP-1 and TSP-2 (1 : 100; Chemicon, Euromedex, Souffelweyersheim, France), anti-CD38 (1 : 100; Santa Cruz Biotechnology), anti-Akt and p-Akt (1 : 500; R&D Systems, Minneapolis, MN, USA), anti-arginase-1 (Arg-1, 1 : 500; R&D Systems, Minneapolis, MN, USA), anti-formyl peptide receptor 2 (Fpr-2, 1 : 500; R&D Systems, Minneapolis, MN, USA), anti-active caspase-3 (1 : 100; Santa Cruz Biotechnology, Santa Cruz, CA), and anti-early growth response protein 2 (EGR2, 1 : 200; Cayman Chemical, Ann Arbor, MI, USA), as described previously [12 (link)]. Omitting primary antibody controls was used to confirm the specificity of staining signals. Five areas from each slide were randomly selected for image acquisition under 200x magnification. The red immunoreactive signal indicated expression of each specific protein, while the green signal indicated TSP-1 and TSP-2 expression; the Hoechst 33342 blue signal indicated nuclei staining.