Roti quant assay

Activities of the effector Caspases-3 and 7, which play a central role in apoptotic events were evaluated in intestinal tissue samples before and after perfusion using rhodamine based fluorometric assays (Apo-One homogeneous Caspase-3/7 assay, Promega Corporation, Madison, WI, USA). Treatment of the samples and evaluation of Caspase-3/7 activity were done on the basis of the manufacturer’s protocol using a fluorescence ELISA reader (Tecan, Crailsheim, Germany) in combination with the Magellan software v1.1. Protein extraction was performed by using RIPA buffer containing 150 mM sodium chloride, 1 % NP-40, 1 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate (SDS) and 50 mM Tris–HCl (pH 7.6; all from Sigma-Aldrich, Hamburg, Germany). The protein concentrations were determined with Roti^®-Quant assays (Carl Roth, Karlsruhe, Germany).

Concentrations of protein samples were determined with Roti-Quant assays (Carl Roth, Karlsruhe, Germany). 20 μg of total protein were mixed with 4× Laemmli buffer (8% SDS, 40% glycerol, 20% 2-mercaptoethanol, 0.008% bromophenol blue, 0.25 M Tris–HCl, all from Sigma-Aldrich) and incubated for 3 min at 95 °C. Samples were separated by 8% SDS-PAGE and transferred to a PVDF membrane (Amersham Pharmacia Biotech, Piscataway, USA). After 2 h of blocking with TBST buffer containing 3% of bovine serum albumin (Carl Roth) at room temperature, the membranes were incubated overnight at 4 °C with specific primary antibodies, anti-CCL26 (1:100; R&D systems, Minneapolis, USA), and anti-RAGE (1:4000; Genetex, Irvine, USA). Signals from peroxidase-conjugated secondary antibodies (anti-rabbit, Cell Signaling Technology, 1:20,000) were detected using the ECL kit (ECL-Plus Western blotting Detection Reagents, Amersham Pharmacia Biotech). Protein bands on the membranes were visualized with the western blot imaging system Fusion FX (Vilber, Collégien, France) and intensities of the respective protein bands were analyzed using the ImageJ software 1.41 (NIH).

Activities of the effector Caspases-3 and -7, which play a central role in apoptotic events were evaluated in luminal effluent samples before and after perfusion using rhodamine based fluorometric assays (Apo-One homogeneous Caspase-3/7 assay, Promega Corporation, Madison, WI, USA). Treatment of the samples and evaluation of Caspase-3/7 activity were done based on the manufacturer’s protocol using a fluorescence ELISA reader (Tecan, Crailsheim, Austria) in combination with the Magellan software v1.1. Protein extraction was performed with RIPA buffer containing 150 mM sodium chloride, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) and 50 mM Tris-HCl (pH 7.6; all from Sigma-Aldrich, Munich, Germany). Protein concentrations were determined with Roti®-Quant assays (Carl Roth, Karlsruhe, Germany) [13 (link)]. To visualize apoptotic cell nucleus in intestinal tissue sections, Tunel staining was performed by using a apoptosis detection kit (ApopTag Peroxidase In Situ, Merk, Darmstadt, Germany) and DAB substrate (Liquid DAB + Substrate Chromogen System, Dako, CA, USA) as described by the manufacturer.