Journal of Immunological Methods, 12 (1976) 117--124 117 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands PREPARATION AND USE IN IMMUNOHISTOLOGY OF ANTIBODIES SPECIFIC FOR TYPE I AND TYPE III COLLAGEN AND PROCOLLAGEN HANS NOWACK, STEFFEN GAY, GEORG WICK *, UDO BECKER and RUPERT TIMPL Max-Planck-lnstitut fiir Biochemie, A bt. Bindegewebsforschung, Martinsreid, Germany, and * Institut fiir allgemeine und experimentelle Pathologie, Universita't Innsbruck, Austria (Received 23 February 1976, accepted 1 March 1976) Antibodies to bovine type I and type III collagen and their precursor form procollagen were produced in rabbits and rendered specific for the immunizing antigen by immunoad- sorption. These purified antibodies showed distinct immunofluorescence staining on frozen sections of both bovine and human connective tissue at concentrations as low as 1--10 ug/ml. Antibodies to type III collagen and procollagen reacted with reticulin in liver and spleen, with fascicles around tendons and with the upper portion of the dermis. Antibodies to type I collagen and procollagen reacted with skin and fiber bundles in tendon but did not stain reticulin. No reaction was observed with cartilage collagen or with kidney glomerular basement membrane. INTRODUCTION The body contains at least four quite distinct collagens (Fietzek and Kiihn, 1976; Martin et al., 1975). Each collagen is formed from a larger precursor, procollagen. In the case of type I collagen, it is known that the precursor has extra peptide sequences at both the NH2 and COOH terminal ends of each s-chain in the molecule (Tanzer et al., 1974; Byers et al., 1975; Fessler et al., 1975). The structures of the precursor forms of type II and III collagen are thought to be analogous. Antibodies can be raised against various antigenic sites in collagen and procollagen including helical and non-helical determi- nants in collagen and precursor-specific sites in procollagen {Furthmayr and Timpl, 1976; Timpl, 1976}. Antisera prepared to one of the genetically dis- tinct collagens possessed very limited ability to react with the other collagen types (Hahn et al., 1974; Becker et al., 1976). Antibodies purified from those antisera have already proved to be valuable reagents for studying bio- logical as well as pathological materials (Olsen and Prockop, 1974; Wick et al., 1975, 1976a; Gay et al., 1975a, b; Miiller et al., 1975; von der Mark et al., 1976), using electronmicroscopical or immunofluorescence techniques. The present study describes the purification of antibodies to type I and type