Bond max automated staining system

Formalin-fixed and paraffin-embedded porcine sling and clasp muscle fibers were prepared for IHC staining. The IHC stains were performed using standard reagents and techniques on a BOND-MAX Automated Staining System (Leica Microsystems). Briefly, the deparaffinized and rehydrated sections were subjected to heat-induced antigen restoration with bond epitope retrieval solution 1 (citrate-based pH 6.0 solution, Leica Microsystems). The staining procedure involved a peroxidase block with 3% hydrogen peroxide for 5 min, incubation with a 1:300 dilution of rabbit neuromedin B receptor polyclonal antibody, LS-A825 (Lifespan Biosciences, Seattle, WA, USA) for BB₁, a 1:300 dilution of rabbit GRPR polyclonal antibody (Abnova, Taipei, Taiwan) for BB₂, or a 1:200 dilution of rabbit anti-Brs3/bombesin receptor 3 polyclonal antibody (Bioss, MA, USA) for BB₃ for 30 min at room temperature. Then, the samples were subsequently incubated with an anti-rabbit horseradish peroxidase polymer for 10 min at room temperature. The samples were then treated with a chromogen, 3,3′-diaminobenzidine tetrahydrochloride (DAB), for 10 min at room temperature and counterstained with hematoxylin for 5 min. The sling and clasp muscle sections were stained with normal rabbit IgG at equimolar concentrations as negative controls.