Mouse anti vimentin

In short, the cells were washed with PBS buffer and fixed using 4% paraformaldehyde for 30min at RT. After permeabilizing in PBS containing 0.1% Triton X-100, the cells were blocked with 1% bovine serum albumin (BSA) for 1h. Then, the cells were incubated with primary antibodies and FITC-conjugated antibody (1:100; Cell Signaling Technology, Danvers, MA, USA). The CytoPainter Phalloidin-iFluor 488 reagent (1:100; Abcam, Cambridge, MA, USA) was used to label microfilaments (MFs) for 30min. Then, Hoechst33342 (1:1000; Abcam, Cambridge) was used to stain the cell nuclei for 30min. The images were taken using Zeiss LSM 800 microscope (LSM800, Zeiss, Germany). The sources of the primary antibodies are as follows: rabbit anti-β-tubulin antibody (1:50; Abcam), rabbit anti-Ki67 antibody (1:50; Abcam) and mouse anti-Vimentin (1:50; Abcam).

Western blotting was performed using standard procedures. Briefly, protein samples (25mg) were separated on polyacrylamide-SDS gels and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore). Subsequently, membranes were blocked and incubated with mouse anti-CD81 (1:1000, Abcam), mouse anti-CD63 (1:1000, Abcam), mouse anti-HIF-1α (1:1500, ProteinTech Group, Inc.), mouse HIF-2α (1:1500, ProteinTech Group, Inc.), rabbit anti-PTEN (1500, Abcam), rabbit anti-E-cadherin (1:1000, Abcam), mouse anti-vimentin (1:1000, Abcam), rabbit anti-mTOR (1:1000, Abcam), rabbit anti-AKT (1:1000, Abcam), rabbit anti-pAKT (1:1000, Abcam), and rabbit anti-tubulin (1:1000 Abcam) antibodies. Anti-rabbit or anti-mouse IgG was used as the secondary antibody (1:8000). The signal intensity was evaluated using Quantity One 4.4.0 software.

Optic nerve sections or whole retinas were washed in PBS (3 × 5 min) and then incubated in blocking solution (5% donkey serum, 0.5% Triton X-100, and 1% bovine serum albumin in PBS) for 1 h at room temperature (RT), followed by incubation in primary antibodies either overnight (optic nerves) or for 3–5 d (retinas), always at 4°C. The primary antibodies used were: rabbit anti-GFAP (1:2,000; Abcam), mouse anti-SMI32 (1:400; Covance), rabbit anti-S100β (1:200; Abcam), mouse anti-vimentin (1:100; Abcam), rabbit anti–βIII-tubulin (1:200; Cell Signaling Technology), mouse anti-GFAP (1:400; Sigma-Aldrich), mouse anti-pSTAT3 (1:100; Cell Signaling Technology), and mouse anti-STAT3 (1:2,000; Cell Signaling Technology). The next day, tissue were washed in PBS (3 × 5 min) and incubated with secondary antibodies conjugated to rhodamine (1:200; donkey anti–rabbit; Jackson ImmunoResearch Laboratories, Inc.) or FITC (1:400; donkey anti–mouse; Jackson ImmunoResearch Laboratories, Inc.) for 2 h (optic nerves) or 3 d (retinas) at RT. Optic nerve sections were washed in PBS (3 × 5 min) and mounted in ProLong Gold Antifade medium (Thermo Fisher Scientific). Retinas were incubated with the nuclear dye DAPI for 20 min, washed in PBS (3 × 5 min), and mounted in Vectashield (Vector Laboratories).