In the present study the effects of contaminating proteinase activities in commercially available preparations of chondroitinases and keratanase were tested upon the histochemical results obtained by their digestion procedures. Tissue sections from the ear and tracheal cartilages, aorta, umbilical cord and cornea of mammals were subjected to staining procedures after prior digestion with the enzyme solutions with or without proteinase inhibitors.In a substantial number of tissue sections examined, the addition of proteinase inhibitors to the enzyme solutions appeared to suppress the decline in intensity of the staining reaction induced by the enzyme digestions.These results are taken to indicate that the commercially available preparations of chondroitinases and keratanase contain significant amounts of contaminating proteinases. When these enzyme preparations are employed as histochemical reagents the addition of proteinase inhibitors to the enzyme solutions is indispensable for precise localization of the enzyme substrates in tissues.In the histochemistry of mucopolysaccharides, several enzyme preparations have been employed as histochemical reagents, such as testicular hyaluronidase (5,(15)(16)(17)24), Streptomyces hyaluronidase (1,12,16,17,19,20,24), chondroitinases (1, 2, 18,21,22) and keratanase (25). As a result of histochemical studies using these enzymes, the localizations of their substrates have been precisely determined in a variety of connective tissues. Of these commercially available enzymes, however, preparations of chondroitinases and keratanase were biochemically shown to contain significant amounts of contaminating proteinases (11), and their proteolytic effect on proteoglycans was shown to be abolished by the addition of appropriate proteinase inhibitors to the enzyme solutions employed (10). Thus, it seems desirable to check the effects of such contaminating proteinase activities on the histochemical results obtained by combined enzyme digestion and staining procedures.In the present study, attempts have been made to examine the effects of added proteinase inhibitors in chondroitinase and keratanase solutions for histochemical digestions upon the staining reactions of the tissue sections containing the substrates of the enzymes. The results obtained reveal that the addition of proteinase inhibitors to the enzyme solutions is indispensable for precise histochemical localization of the substrates of the enzymes in tissues.
MATERIALS AND METHODS
PREPARATION OF TISSUE SECTIONS:In the present study, organs which are known to contain substantial amounts of the substrates of chondroitinase ABC and AC and keratanase from different mammalian species were employed. Thus, ear cartilage (rabbit), tracheal cartilage (rabbit and rat), aorta (monkey and pig), umbilical cord (human being and pig) and cornea (rabbit and rat) were used. Tissue specimens from these organs were fixed at 4°C for 24 to 48 hr in either 1 % cetylpyridinium chloride (CPC) in 10% formalin (7, 15) or 2 % calcium acetate in 10% forma...