Immunohistochemistry (IHC) remains a cornerstone method in diagnostic pathology; however, the concurrent visualization of multiple antibodies using various chromogens is typically labor-intensive, technically challenging, and relatively costly. To simplify mast cell (MC) recognition during immunohistochemical evaluation of membrane or nuclear antigens, we introduce a novel staining protocol combining IHC with toluidine blue as a counterstain. This approach was applied to assess c-kit, Ki67, and cannabinoid receptor 2 expression across several cases of canine cutaneous mast cell tumors (MCTs), mastocytosis, and atopic dermatitis. Our findings indicate that this dual-staining approach—though applicable only to non-cytoplasmic markers and less effective in poorly differentiated MCTs where MC metachromasia is indistinct—offers practical value in analyzing membranous and nuclear markers in canine skin diseases, especially those containing sparse MC populations. It aids in differentiating neoplastic MCs from infiltrating inflammatory cells during Ki67 index determination and serves as a valuable tool for investigating new molecular markers in both veterinary and human mast cell–related conditions.
