The AGS cell samples were processed using standard procedures [27 (link)]. The slides were consecutively incubated with 1) anti-GPR39 or anti-obestatin in Dako ChemMate antibody diluent (Dako, Glostrup, DK); 2) EnVision™ peroxidase rabbit (Dako, Carpinteria, CA, US), which was used as the detection system; and 3) 3,3′-diaminobenzidine-tetrahydrochloride (Dako Liquid DAB + Substrate-chromogen system). The cells were faintly counterstained with Harris hematoxylin solution (HHS). The preadsorption control was performed by applying the primary antibody plus obestatin or the GPR39 control peptide to the positive samples.
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