Ab8979

To detect GFP-labeled BMSC, paraffin-embedded sections were twice deparaffinized with xylene for 5 minutes, and rehydrated in a series of graded alcohol solutions (70%–100%). Endogenous peroxidases were inhibited by incubation with 3% H2O2 in PBS buffer. For antigen retrieval, the samples were heated to 98°C–99°C in antigen-retrieval buffer (10 mMTri-sodium citrate, 0.05% Tween 20, pH 6.0) and incubated for 30 minutes in the pressurized vessel. Nonspecific staining was blocked with a mixture of sera in 1.5%PBS for 30 minutes at room temperature, and incubated in the mixture of two primary antibodies in a pair-wise fashion with the mouse monoclonal anti-GFP antibody (SC-9996) at 1:50 dilutions for 1 hour at room temperature. Following incubation with the appropriate fluorescent-conjugated secondary antibodies, the sections were covered with mounting medium containing DAPI (Santa Cruz, Heidelberg, Germany). The cells were investigated under fluorescence microscope (Leica DMI 4000B; Leica Mycrosystems, Wetzlar, Germany). For other immunostainings, the following antibodies were supplied from Abcam (Cambridge, MA, USA): GFAP (ab4674), anti-vimentin antibody (ab8979) BRN3A (ab81213), and rhodopsin (ab3267). The dilution rate of all primary antibodies was 1:100.

Cells were harvested and lysed with RIPA lysis buffer containing protease and phosphatase inhibitor (Thermo Scientific, Beijing, China). Protein concentration was measured using the BCA-kit, and 20 μg of each sample was resolved by SDS-PAGE. The primary antibodies included rabbit anti-human NEDD4 (Abcam, ab14592, UK), mouse anti-human AKT, rabbit anti-human p-AKT (Santa Cruz Biotechnology, sc-5298/135650, Dallas, TX, USA), rabbit anti-human PTEN (Abcam, ab32199, UK); mouse anti-human E-cadherin (Abcam, ab1416, UK), mouse anti-human Vimentin (Abcam, ab8979, UK) and mouse anti-human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Abcam, ab8245, UK). Optical densitometry analysis was performed using Image J software. GAPDH served as a loading control in each case.

Proteins were extracted from clinical tissue samples and culturing cells using the RIPA buffer supplemented with proteinase inhibitor cocktail (Pierce Biotechnology) according to the manufacturer's protocols. Protein concentrations were determined using the BCA Protein Assay Kit (Pierce). Extracted proteins were isolated using 10% SDS‐polyacrylamide gel electrophoresis and then transferred onto the PVDF membranes, followed by 5% nonfat milk blocking. Afterward, the membranes were probed using primary antibodies. The primary antibodies were listed as followed: anti‐CCND1 (1:1000, ab134175; Abcam), anti‐p21 (1:1000, #2947; CST), anti‐Snail (1:1000, ab180714; Abcam), anti‐Vimentin (1:1000, ab8979; Abcam), and anti‐β‐actin (1:500, ab20272; Abcam). Following incubation with HRP‐conjugated secondary antibody, the blots were visualized using the ECL detection system (Amersham).