Envision 3 3 diaminobenzidine dab substrate chromogen

For verification of the sites of occupation, sections from all experimental groups, fixed with NBF and MMC, were treated for 16 h with rabbit hyperimmune serum against infectious larvae of T. spiralis (1 : 5 000), then incubated for 30 min with EnVision TM anti-rabbit polymer conjugated with horseradish peroxidase (Dako) and the peroxidase activity was developed with En Vision TM 3,3'-diaminobenzidine (DAB+) Substrate-Chromogen (Dako). In parallel, negative control specimens were incubated with REAL antibody diluents (Dako) instead of antibody and thereafter with the corresponding peroxidase conjugate. The sections were counterstained with hematoxylin, mounted in acrylic resin and examined with Nikon Eclipse 80ί light microscope (Nikon Engineering Co., Ltd., Yokohama, Japan).
The intracellular staining intensity of the occupied fibres was evaluated semi-quantitatively versus the surrounding non-affected sarcoplasm by two independent observers into two categories: negative (-) and positive (+). The number of invaded fibres was counted in 12 transverse sections of three muscles from each group and their percentage was calculated versus the total number of fibres per section.