Rabbit peroxidase labeled secondary antibodies

Fragments of gastric fundi from the control and hormone-exposed specimens (3 samples for each experimental group) were homogenized in cold lysis buffer (20 mmol /L Tris/HCl (pH 7.4), 10 mmol/L NaCl, 1.5 mmol/L MgCl2, 5 mmol/L EGTA, 2 mmol/L Na2EDTA, mixed with 10× Sigmafast Protease Inhibitor Cocktail tablets). The total protein content was measured spectrophotometrically using the micro-BCA™ Protein Assay Kit (Pierce, IL, USA). Fifty micrograms of total protein was electrophoresed by SDS–PAGE and blotted onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were incubated overnight (O.N.) at 4 °C with rabbit polyclonal anti-nNOS (1:2000; Millipore) and rabbit polyclonal anti-β-actin (1:20,000; Sigma Aldrich, St. Louis, MO, USA), assuming β-actin as a control invariant protein. Specific bands were detected using rabbit peroxidase-labeled secondary antibodies (1:15,000 Vector, Burlingame, CA, USA) and enhanced chemiluminescent substrate. Densitometric analysis of the bands was performed using Scion Image Beta 4.0.2 image analysis software (Scion Corp., Frederick, MD, USA).

Fragments of gastric fundus from the control and the ADPN-treated mice were quickly minced and homogenized with a tissue homogenizer (Ing. Terzano, Milan, Italy) in cold lysis buffer of the following composition: 20 mM Tris/HCl (pH 7.4), 10 mM NaCl, 1.5 mM MgCl₂, 5 mM EGTA, 2 mM Na₂EDTA, added with 10× Sigmafast Protease Inhibitor Cocktail tablets and 100 mM sodium orthovanadate. Total protein content was measured spectrophotometrically using micro-BCA™ Protein Assay Kit (Pierce, IL, USA). Seventy μg of total proteins were electrophoresed by SDS–PAGE and blotted onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal anti-AMPK (1: 1000; Cell Signaling, Danvers, MA, USA), rabbit polyclonal anti phosphoAMPK (Thr172), (pAMPK) (1:1000; Cell Signaling), and rabbit polyclonal anti-β-actin (1:20,000; Sigma Aldrich, Milan, Italy), assuming β-actin as control invariant protein. Specific bands were detected using rabbit peroxidase-labeled secondary antibodies (1:15.000 Vector, Burlingame, CA, USA) and enhanced chemiluminescent substrate. Densitometric analysis of the bands was performed using Scion Image Beta 4.0.2 image analysis software (Scion Corp., Frederick, MD, USA).