Immunohistochemical study of canine mammary gland tumours

Abstract: This study was carried out to determine the phenotype of special dog mammary gland tumours that were grown in nude mice. 26 tumours were examined by the immunohistochemical ABC-Elite protocol. The tumour tissues were labelled with following anti-human antibodies: - AE1/AE3 (pankeratin antibody) labelled epithelial and myoepithelial cells - CD 31 labelled endothelial cells - desmin labelled cross-striated and smooth muscle cells - myosin labelled cross striated muscle cells - neurofilament (NF) labelled nerve cells - osteopontin labelled preosteoblasts, osteoblasts and osteocytes - p63 labelled nuclei of the myoepithelial cells - smooth muscle actin (SMA) labelled the cytoplasm of myoepithelial cells - type I collagen labelled the extracellular matrix in connective tissue and bone - type II collagen labelled the extracellular matrix in cartilage - vimentin labelled fibroblasts, fibrocytes, lipocytes, smooth muscle cells, endothelial cells, nerve cells, macrophages and myoepithelial cells The tumours were also submitted to a double immunolabelling study using p63 and SMA. The study could not give a final conclusion about the origin the tumours. There was still need for more research to answer that question. However, the immunohistochemical technique was analysed in detail, in order to obtain perfect labelings. Initially, all the antibodies were tested on normal dog tissue, to acquire the best working dilutions with the lowest background problems. In the tumours, good results were obtained with these dilutions for the antibodies p63, SMA, vimentin, desmin, NF, AE1/AE3 and CD 31. Except for type I collagen, type II collagen and osteopontin that gave too much unspecific labelling of the mouse connective tissue. Even, when using the Vector® M.O.M. blocking kit, the results were still very difficult to interpretate. The antigen retrieval methods were evaluated for all the antibodies. The antibodies p63, SMA, vimentin, desmin, AE1/AE3, myosin, neurofilament and CD 31 needed the antigen retrieval treatment. The antibodies type I collagen and type II collagen needed the treatment with the enzyme pepsin, while osteopontin did not need any pretreatment at all. The double immunolabelling with p63 and SMA gave excellent results. Different combinations were tried out with different substrates, namely Vector® Nova RED, Vector® DAB and Vector® SG. Vector® methyl green was used as counterstaining, but it interfered with the other substrates, and better results were obtained without this counterstaining.