Histochemie 34, 77--84 (1973) 9 by Springer-Verlag 1973 Fluorescent-Labelled Aprotinin: A New Reagent for the Histochemical Detection of Acid Mucosubstances J. A. Kiernan * Department of Anatomy, University of Cambridge, Cambridge, England R. W. Stoddart Strangeways Research Laboratory, Cambridge, England Received October 10, 1972 Summary. Aprotinin (Trasylol| a polypeptide protease inhibitor from bovine orga~ns, has been labelled with fluorescein isothiocyanate to produce a fluorecent conjugate (FLA). The conjugate h0~s been applied to paraffin sections of rat tissues and has been shown to act as a specific fluorochrome for acidic mucosubstances. There is reason to believe that FLA stains carbohydrates which owe their acidity to the presence of carboxyl groups. The histo- chemical findings are described and discussed and it is also suggested that attachment of aprotinin to extracellular polysaccharides in vivo may limit the therapeutic usefulness of the drug. Introduction Aprotinin is a polypeptide inhibitor of proteoly~ic enzymes extracted from bovine organs. The inhibitor forms complexes with trypsin, chymotrypsin, some kallikreins and some plant proteases (see Vogel, Trautschold and Werle, 1968, for biochemical data). The aprotinin molecule contains four lysine residues which are not important to the inhibitory properties and can, therefore, be labelled without loss of activity by conjugation with fluorescein isothiocyanate. It was at first hoped that the labelled inhibitor might be used to localize proteolytic enzymes in frozen sections of animal tissues, but no evidence of such specificity was obtained. Instead it was observed that the labelled inhibitor principally stained components of tissues known to be rich in acid mucosubstanees. A biochemical study by Stoddart and Kiernan (1973b)has indicated that labelled approtinin forms complexes with those sugars, polysaccharides and glycoproteins which owe their acidity to carboxyl groups. The histochemieal use of the new reagent is described below and followed by a discussion of the possible significance of the findings in relation both to histochemical practice and to the pharmacological properties of aprotinin. Materials and Methods Preparation o/ Reagent. A solution of aprotinin, as marketed for clinical use as Trasylol| was supplied by Bayer Pharmaceuticals Ltd., Haywards Heath, Sussex, England, in ampoules * Present address: Department of Anatomy, Health Sciences Centre, University of Western Ontario, London 72, Ontario, Canada.