Computational protocol: Experimental study of the protective effects of SYVN1 against diabetic retinopathy

Similar protocols

Protocol publication

[…] A flat-mount retinal preparation after FITC-dextran perfusion was used to visualize vascular leakage in mouse retinas (one eye per mouse and three mice per group). Mice were euthanized with sodium pentobarbital (45 mg/kg) by intraperitoneal injection. The mice were placed in the supine position, and 0.3 mL of a high-molecular-weight FITC-conjugated dextran solution (2 × 106; 25 mg/mL; Sigma, USA) was perfused via the superior vena cava. Five minutes after intravital circulation, the eyes were enucleated, immediately placed in paraformaldehyde solution (40 g/L), and fixed for 40 min. The retinas were carefully dissected using forceps and flat-mounted onto glass slides. Retinal vessels were visualized under an Olympus BX51 fluorescence microscope (Olympus America, Center Valley, PA, USA), and retinal vascular leakage was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).The retinal vasculature was also histologically analyzed. Mice were euthanized with an overdose (90 mg/kg) of sodium pentobarbital. The eyes were enucleated and immediately placed in paraformaldehyde solution (40 g/L) for 24 h. The eyes were then dehydrated using a graded ethanol series and embedded in paraffin. The whole eyes were serially sectioned (5-μm thick) along the vertical meridian and stained with hematoxylin and eosin (HE; Hubei BIOS Bio-tech Co.; Ltd.). One eye per mouse and three mice per group were analyzed. Three sections from each eye at the same position were stained for analysis using ImageJ software (National Institutes of Health, Bethesda, MD, USA).Retinal cell apoptosis and DNA damage were measured by the transferase-mediated dUTP nick-end labeling (TUNEL) assay. Mice were euthanized with an overdose of sodium pentobarbital. The eyes were enucleated and immediately placed in paraformaldehyde solution (40 g/L) overnight. The eyes were then dehydrated for 8 h using 30% sucrose solution, and the anterior segment and vitreous body were removed. The eyes were embedded in Tissue-Tek OCT medium (Sakura-Finetek, Japan, Tokyo, Japan) and serially cryo-sectioned (10 μm). One eye per mouse and three mice per group were analyzed. Three sections from each eye at the same position were labeled using a TUNEL kit according to the manufacturer’s instructions (Roche, Mannheim, Germany). The labeled retinas were visualized by confocal laser-scanning microscopy (Nikon Eclipse Ti-SR; Japan) and analyzed using ImageJ software (National Institutes of Health).Retinal tissue and frozen sections were obtained as described above. Immunofluorescent labeling was carried out as previously described, with minor modifications. Immunofluorescent double labeling was performed for VEGF and CHOP after staining sections for SYVN1. The following primary antibodies were used: anti- SYVN1 (1:100; bs-0679R; Biosynthesis Biotechnology, Beijing, China), anti-VEGF (1:200; ab1316; Abcam, USA), and anti-CHOP (1:150; BS1527; Bioworld). The sections were incubated in the primary antibody solution at 4 °C overnight, washed, and labeled with Cy3- or FITC-conjugated secondary antibodies (1:100; BA1105, BA1031; Boster, Wuhan, China). The nuclei were stained with DAPI. The labeled sections were visualized by confocal laser-scanning microscopy (Nikon Eclipse Ti-SR; Nikon, Tokyo, Japan). [...] Normally distributed data were statistically analyzed with the independent two-sample t-test or one-way ANOVA using SPSS Statistics15.0 software (SPSS Inc., Chicago, IL, USA). Data were presented as the mean ± standard deviation (SD). Differences with p-values of less than 0.05 were considered statistically significant. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Diabetes Mellitus, Retinal Diseases, Machado-Joseph Disease
Chemicals Streptozocin