Anti nse

Protein concentration was measured using the bicinchoninic acid (BCA) method (Thermo Scientific). Subsequently, 20 μg of cell lysate was denatured with sample buffer (62.5 mM Tris–HCl pH6.8, 2% (v/v) SDS, 50 mM DTT, 10% (v/v) glycerol), subjected to 4%–20% gradient SDS-PAGE, and transferred onto nitrocellulose membranes (BioRad). The membranes were blocked for 1 h with 5% (w/v) dried skimmed milk in TBS-T buffer (25 mM Tris, 150 mM NaCl, and 0.1% (v/v) Tween-20, pH 7.4), and incubated with the following primary antibodies: mouse anti-GFAP (Sigma-Aldrich; 1:50,000), rabbit anti-Iba1 (Wako; 1:10,000), mouse anti-β-actin (Sigma-Aldrich; 1:20,000), anti-NSE (1:1000, Dako), or anti-GAPDH (1:40000, GeneTex), at 4°C overnight. Then, membranes were incubated with the corresponding HRP-coupled secondary antibodies (Jackson Immunoresearch; 1:10,000) for 1 h at room temperature, and the bands were visualized using the Super Signal West Pico chemiluminescent substrate (Thermo Scientific). The immunoreactive signals were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Iba1 and GFAP values were normalized to β-ACTIN values. NSE results, due to molecular weight, were normalized to GAPDH values.

CgA-CreERT2 mice were crossed with Rosa-CAG-LSL-ZsGreen1 Cre-reporter mice. The offspring were intraperitoneally injected with tamoxifen (1 mg/body) dissolved in corn oil once daily for 3 days. The duodenum, pancreas, and brain were resected and fixed with 10% formalin. Paraffin-embedded tissue sections were immunostained with anti-chromogranin A, anti-insulin, and anti-NSE (DAKO, Glostrup, Denmark) as described [17 (link)]. The sections were visualized with fluorescently (Alexa 555) labeled secondary antibodies (Life Technologies) on a BZ9000 microscope (KEYENCE, Tokyo, Japan).