Amersham biosciences ecl western blotting detection reagent

The procedures used for the immunoblotting assay were previously described [18 (link)]. Cells were lysed in a buffer containing 150 mm NaCl, 20 mm Tris/HCl [pH 7.5], 1% Nonidet P‐40, 1 mm EGTA, 5 mm EDTA, phosphatase inhibitors and protease inhibitor cocktails. Protein concentration was measured using BCA protein assay reagent (Thermo Fisher Scientific, Waltham, MA, USA). The collected proteins were separated by SDS/PAGE and transferred to polyvinylidene difluoride membranes (Pall, Glen, Cove, NY, USA). The blots were incubated at room temperature for 1 h. The working dilution of primary and HRP‐conjugated secondary (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) antibodies was 1 : 1000 and 1 : 10 000, respectively. Proteins were visualized using Amersham Biosciences ECL Western blotting detection reagent (GE Healthcare, Chicago, IL, USA). All data were obtained from the image files acquired using a LAS‐4000 mini luminoimage analyzer (Fujifilm, Tokyo, Japan).

Western blotting analyses was performed as described in our previous publication [24] (link). Briefly, within two minutes after enucleation, the retinas were quickly isolated and shock frozen at −80°C. Then, the retina was sonicated in RIPA buffer (Santa Cruz Biotech Inc. Dallas, TX). The protein concentrations were measured using a BCA protein assay. Then, the proteins were transferred to nitrocellulose membranes (Millipore Corp, Billerica, MA). After blocking with 5% BSA for 1 hour at room temperature, the membranes were incubated with primary antibodies at 4°C overnight, including goat polyclonal antibody against ChAT (Millipore Corp, Billerica, MA), rabbit polyclonal antibody against HO-1 (Stressgen Biotech Inc., Philadelphia, PA.), and rabbit polyclonal anti–β-actin (Sigma-Aldrich Corp). Next, the membranes were washed and incubated with horseradish peroxidase-conjugated an anti–goat secondary antibody and an anti–rabbit secondary antibody (PerkinElmer, Inc., Wellesley, MA) for 1 h at room temperature. The Amersham Biosciences ECL Western blotting detection reagent (GE Healthcare Life Science, Uppsala, Sweden) was used for signal detection according to the manufacturer's protocol. The optical density value (OD) of each band was measured using software Image J.