THE IMPACT OF FIXATIVES ON THE BINDING OF LECTINS TO N-ACETYL-GLUCOSAMINE RESIDUES OF HUMAN SYNCYTIOTROPHOBLAST - A QUANTITATIVE HISTOCHEMICAL-STUDY

We describe a quantitative histochemical method for demonstration of f ive N-acetyl-glucosamine binding lectins in the syncytiotrophoblast of human term placenta, The method employs biotinylated lectins and alka line phosphatase-conjugated avidin. The alkaline phosphatase activity is detected by using 5-bromo-4-chloro-indoxyl phosphate as the substra te and nitroblue tetrazolium as the capture agent. The effect of 13 fi xative solutions on specific lectin binding and nonspecific background staining was quantified by microspectrophotometry. Acid fixatives or fixatives containing mercuric chloride, e.g., Carnoy's and Zenker's fi xatives, gave intense specific lectin binding and low background stain ing. Glutaraldehyde, carbodiimide, and ethanol resulted in low specifi c lectin binding and a very high background staining that was mainly d ue to endogenous placental alkaline phosphatase, Lectin binding to N-a cetyl-galactosamine, mannose, galactose, and fucose was also significa ntly higher in sections from tissues fixed in an acid fixative compare d with a neutral buffered fixative, Unfixed cryosections revealed a co nsiderably lower degree of specific lectin binding compared with secti ons from fixed tissues, The activity of endogenous placental alkaline phosphatase was inhibited dose-dependently by mercuric chloride and de creased with L-phenylalanine concentration over the range of 7.8 x 10( -4) M to 5 x 10(-2) M, after which there was no further inhibition, Ca lf intestinal-type alkaline phosphatase conjugated to avidin was not i nhibited by 5 x 10(-2) M L-phenylalanine. Endogenous placental biotin did not contribute significantly to background staining. Despite the h igh level of placental alkaline phsophatase, the intestinal-type alkal ine phosphatase can be used as a marker enzyme in the sensitive ABC te chnique, provided that the nonspecific background is measured and subt racted. Moreover, it is advisable to use an acid- and/or mercuric chlo ride-containing fixative and to add L-phenylalanine during incubation steps.