A Double Histochemical/Immunohistochemical Staining for the Identification of Canine Mast Cells in Light Microscopy

Abstract

Immunohistochemistry (IHC) is a widely used technique in diagnostic pathology, but the simultaneous analysis of more than one antibody at a time with different chromogens is rather complex, time-consuming, and quite expensive. In order to facilitate the identification of mast cells (MCs) during immunohistochemical analysis of membrane and/or nuclear markers, we propose a new staining method that includes the association of IHC and toluidine blue as a counterstain. To achieve this goal, we tested c-kit, Ki67, and cannabinoid receptor 2 on several cases of cutaneous canine mast cell tumors (MCTs), cutaneous mastocytosis, and atopic dermatitis. The results obtained show how this double staining technique, although limited to non-cytoplasmic markers and of little use in poorly differentiated MCTs in which MC metachromasia is hard to see, can be used during the evaluation of nuclear and/or membranous immunohistochemical markers in all canine cutaneous disorders, especially if characterized by the presence of a low number of MCs. It can help to evaluate those MCTs in which neoplastic MCs must be clearly distinguished from inflammatory cells that can infiltrate the tumor itself, in facilitating the calculation of the Ki67 index. Moreover, it can be used to study the expression of new markers in both animal and human tissues containing MCs and in MC disorders.

Cannabinoid receptors in the inflammatory cells of canine atopic dermatitis

“…The expression of cannabinoid and cannabinoid-related receptors has already been shown in the MCs of humans (64, 68-70), rodents (13,71), dogs (43,44), and cats (50), and in the MC cell line (72).…”

“…Unexpectedly, some difficulties in MC identification were encountered, and it was necessary to test more anti-tryptase antibodies. The only antibody, among those tested, capable of effectively identifying MCs in the cryosections was the rabbit anti-tryptase antibody (PAB070Ca01), raised against the tryptase of dogs ( 43 ); its specificity was also tested in the current study by combining immunohistochemical staining in association with toluidine blue as a counterstain ( 44 ) ( Supplementary Figure 2 ). In the present study, the rabbit anti-tryptase antibody was used in co-localization with the mouse anti-CB2R antibody.…”

“…A recent study has demonstrated that cannabidiol (CBD), a non-psychotropic phytocannabinoid showing numerous health-related benefits, including anti-inflammatory and anti-anxiety properties ( 41 , 42 ), may be useful in dogs with AD ( 1 ). Despite these promising clinical studies, there are still few studies dedicated to the histological localization of cannabinoid receptors in the canine inflammatory cells ( 43 , 44 ). It is evident that knowing the cellular distribution of specific receptors is fundamental to understanding the action of a drug.…”