Ab1316

To investigate the potential roles of lncRNA TDRG1 and VEGF in angiogenesis in the progression of DR, immunofluorescence staining of VEGF and lncRNA TDRG1 was performed using paraffin‐embedded sections or cultured HRECs on poly-lysine‐coated glass coverslips under hyperglycemic conditions. The slides were rinsed, and the cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X‐100 in PBS for 15 min at room temperature, and blocked (1% bovine serum albumin in PBS) for 1 h at 37°C. Subsequently, the sections were treated with primary rabbit polyclonal anti‐VEGF antibody (1:100, Abcam #ab1316, USA), and RNA fluorescence in situ hybridization of lncRNA TDRG1-FISH (1:100, Servicebio, China) was performed at 4°C overnight. Slides were then incubated with Cy3‐conjugated goat anti‐rabbit or goat anti‐mouse IgG DyLight 488‐conjugated secondary antibodies (1:500, CST Signaling, USA) for 1 h at 37°C. Nuclei were stained with DAPI (1:5,000 diluted in PBS; Thermo, IL, USA). The slides were observed under a confocal laser scanning microscope (Leica, Germany). The primer sequence of lncRNA TDRG1-FISH was: 5′-CCTTGCCAGGTAAGTGAAAGTGCGCTCCG-3’. The mean fluorescence intensities were calculated by ImageJ software. Five images for each group in the three independent experiments were evaluated. Specifically, the cell number was included in the calculation process.

Extracted protein samples were boiled for 5 min and separated by electrophoresis (Bio-Rad, Richmond, CA) in 12% SDS-PAGE gel before electroblotted (Bio-Rad) onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA) [11 (link)]. After polyvinylidene fluoride membranes were blotted with Tris buffer containing 5% fat-free dry milk and 0.05% Tween-20 (TBST; 0.05% Tween 20, 100 mmol/L of Tris-HCl, and 150 mmol/L of NaCl, pH 7.5) for 1 h at 25°C, they were rinsed in TBST for 4 times and incubated overnight at 4°C with primary antibodies, which were mouse polyclonal GAPDH (Abcom, ab22556), rabbit polyclonal HIF-1α (Abcam, ab114977), mouse monoclonal VEGF (Abcam, ab1316) and rabbit polyclonal VEGFR1 (Abcam, ab2350) with 1:3000, 1:2000, 1:2000 and 1:2000 dilutions, respectively. The membranes were washed in the same manner as described above and incubated with the horseradish peroxidase-conjugated secondary antibody (1:5,000; Sigma) for 40 min. Then, the membranes were washed 6 times for 5 min in TBST and the blots were detected using Lumi-light Western Blotting substrates (Biofuture, Beijing, China) and analyzed with the Imaging Analysis Software of National Institutes of Health (Bethesda, MD).

Formalin-fixed paraffin sections were deparaffinized in xylene and rehydrated through an ethanol series. The sections were then immersed in 0.3% hydrogen peroxidase for 15 min to deplete endogenous peroxidase and exposed to high pressure to activate their antigenicity. Sections were then incubated with blocking solution (PBS, 3% of BSA, 0.1% Triton-X 100) at room temperature for 30 minutes. The primary antibody of TLR4 (1:20, ab22048; Abcam), MD2 (ab24182; Abcam), Myd88 (ab2068; Abcam), NF-κB (ab7970; Abcam), IL-6 (ab6672; Abcam), TGF-β (ab66043; Abcam), VEGF (ab1316; Abcam), EGF (ab115562; Abcam), and MMP2 (ab86607; Abcam) were added and incubated at 4 °C overnight. Sections were washed with PBS, incubated with FITC–conjugated goat anti-mouse secondary antibody at room temperature for 1 hour (1:400), and washed three times with PBS at room temperature for 10 minutes. Images were obtained with a fluorescence microscope (OLYMPUS DP70; Olympus Co., Tokyo, Japan).

PIMC (10⁴ cells per well) were seeded in 8-well Nunc Lab-Tek Chamber Slides (Invitrogen, Carlsbad, CA, USA). After reaching the 50% confluence cells were washed with IMDM, fixed with 3.7% paraformaldehyde in IMDM, and 0.2% Nonidet P-40 was used to permeabilize the cells. Slides were incubated for 2 hours at 37°С with primary antibodies against α-SMA (ab7817), aggrecan (ab36861), fibronectin (ab45688), СD34 (ab81289), VEGF-A (ab1316) (Abcam, Cambridge, UK), CD105 (M352701-2), CD31 (PECAM1) (M082301-2) (Dako, Agilent, Santa Clara, CA, USA), CD14 (2109035) (Sony Biotechnology Inc., San Jose, CA, USA), then incubated for 1 hour at 37°С with Alexa 488-conjugated secondary antibodies (ab150077 and ab1500117, Abcam, Cambridge, UK). Samples were stained with rhodamine phalloidin (Sigma-Aldrich, Saint Louis, MO, USA) for F-actin, and glass slides were covered with the Prolong Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA, USA) for nuclei staining, mounted with coverslips and analyzed using confocal laser scanning microscopes LSM 510 UV Meta or LSM 780 NLO (Carl Zeiss, Jena, Germany) using three laser lines at 405 nm (to detect cell nuclei stained with DAPI), 543 nm (to detect actin microfilaments stained with phalloidin-TRITC), and 488 nm (to detect Alexa Fluor 488 secondary antibodies).

Cell lysate was collected using RIPA lysis buffer. The cell protein mass of each lysate was determined by BCA Protein Assay Reagent (Pierce, IL). Proteins were separated using 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to membranes (0.22 μm, Sigma) and incubated with primary antibodies at 4°C overnight. The primary antibodies are as follows: antibodies against VEGFA (1/1000; ab1316, Abcam), TGFβ1 (1/1000; ab215715, Abcam), Angiopoietin-1 (1/20000; ab183701, Abcam), ZO-1 (1/1000; ab216880, Abcam), Occludin (1/1000; ab216327, Abcam), Claudin-5 (1/1000; ab131259, Abcam), G3BP2 (1/2000; ab86135, Abcam), p-p38 (1/1000; ab195049, Abcam), p38 (1/2000; ab170099, Abcam), p-p53 (1/2000; ab33889, Abcam), p53 (1/1000; ab26, Abcam), and GAPDH (1/500; ab8245, Abcam). Next, the membranes were incubated with HRP-conjugated secondary antibody IgG (1/2000; ab7090, Abcam) at room temperature for 2 h. GAPDH antibody served as a negative control. At last, the protein bands were visualized by an ECL Western Blotting Substrate Kit (ab65623, Abcam) and quantified by the ImageJ software.

The antibody against inhibin‐α (R1, ab14087, Abcam, Cambridge, United Kingdom) was used for immunohistochemistry and immunofluorescence analyses (dilution at 1:200). The antibody against inhibin‐α (4A2F2, ab47720, Abcam) was used for immunoblotting (dilution at 1:500) and flow cytometry analyses (1 µg for 10⁶ cells.). The antibody against ALDH1A1 (D9Q8E, 54135, Cell Signaling Technology, Inc., Danvers) was used for immunohistochemistry (dilution at 1:2000) and immunoblotting (dilution at 1:1000) analyses. Antibody for immunohistochemistry against Ki‐67 (MIB‐1, M7240, dilution at 1:100) was obtained from Dako/Agilent Technologies, Inc. (Santa Clara). Antibody for immunoblotting against β‐actin (horseradish peroxidase [HRP] conjugate) (13E5, 5125, dilution at 1:1000) was obtained from Cell Signaling Technology, Inc. The antibody against vascular endothelial growth factor (VEGF)‐A (VG‐1, ab1316, Abcam) was used for immunohistochemistry and immunoblotting (dilution at 1:200). The antibody against VEGF‐C (9/E7, ab106512, Abcam) was used for immunohistochemistry and immunoblotting (dilution at 1:400).