Envision peroxidase rabbit

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.

Paraffin sections of feline kidneys were prepared as described previously (Miyazaki et al., 2003). Endogenous peroxidase was blocked with 0.3% H₂O₂ in methanol for the detection of HRP activity. Deparaffinized sections were incubated with 10% goat serum in PBS at room temperature for 1 hour and then with anti‐human L‐FABP monoclonal antibody (clone 2, CMIC Holdings, diluted 1:200) at room temperature for 1 hour. After treatment with HRP‐labeled polymer‐conjugated anti‐mouse IgG antibody (EnVision+, peroxidase, rabbit; Dako, Glostrup, Denmark) for 30 minutes at room temperature. The sections were treated with diaminobenzidine to visualize the antigen‐antibody reaction. Finally, the sections were counterstained with hematoxylin and observed under a light microscope.