Computational protocol: Nestin Expression in the Adult Mouse Retina with Pharmaceutically Induced Retinal Degeneration

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Protocol publication

[…] After the careful dissection of the retinas from the eye cups, the retinal tissue was flash frozen in −80°C liquid nitrogen until samples at all time points were collected. A 100 µL ice-cold extraction buffer containing 10 mM of Tris-HCl (pH = 7.5), 5 mM of ethylenediaminetetraacetic acid (EDTA), 200 mM of NaCl, 1 mM of phenylmethylsulfonyl fluoride, 1 µg/mL of leupeptin, and 28 µg/mL of aprotinin was added to both retinal tissues from one mouse. The retinal samples were sonicated and centrifuged at 12,000 rpm for 30 minutes at 4°C. The supernatant was collected, and protein concentrations were measured according to the manufacturer's instructions (BCA Protein Assay Kit; Thermo Scientific, Rockford, IL, USA). Proteins (30 µg of each sample) were fractionated on 8% polyacrylamide precast gels (Bio-Rad, Cambridge, MA, USA), and transferred to a polyvinylidene difluoride (PVDF) membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). The membranes were blocked for 2 hours at room temperature in Tris-buffered saline (TBS) containing 5% skim milk and were then incubated with nestin (mouse monoclonal, 1:5,000; Millipore) or GFAP antibody (rabbit polyclonal, 1:1,000; Sigma-Aldrich) overnight at 4°C. The membranes were finally incubated with peroxidase-conjugated anti-mouse or anti-rabbit IgG (Sigma-Aldrich) for 2 hours at room temperature. Enhanced chemoluminescence (ECL) detection of nestin or GFAP was performed according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ, USA). The expression of those markers was used to quantify and compare them with each other using ImageJ software, version 1.47c (Image Processing and Analysis in Java; National Institutes of Health, Hamilton, MO, USA). [...] The Mann-Whitney U test was used to assess differences in cell counts at the five designated time points after MNU administration. Statistical analysis was conducted using SPSS Statistics Version 21 (IBM, Somers, NY, USA). All tests were two-tailed, and differences having P values of less than 0.05 were considered statistically significant. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Retinal Degeneration
Chemicals Calcium, Methylnitrosourea