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 five N-acetyl-glucosamine binding lectins in the syncytiotrophoblast of human term placenta. The method employs biotinylated lectins and alkaline phosphatase-conjugated avidin. The alkaline phosphatase activity is detected by using 5-bromo-4-chloro-indoxyl phosphate as the substrate and nitroblue tetrazolium as the capture agent. The effect of 13 fixative 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 fixatives, gave intense specific lectin binding and low background staining. Glutaraldehyde, carbodiimide, and ethanol resulted in low specific lectin binding and a very high background staining that was mainly due to endogenous placental alkaline phosphatase. Lectin binding to N-acetyl-galactosamine, mannose, galactose, and fucose was also significantly higher in sections from tissues fixed in an acid fixative compared with a neutral buffered fixative. Unfixed cryosections revealed a considerably lower degree of specific lectin binding compared with sections from fixed tissues. The activity of endogenous placental alkaline phosphatase was inhibited dose-dependently by mercuric chloride and decreased 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. Calf intestinal-type alkaline phosphatase conjugated to avidin was not inhibited by 5 x 10(-2) M L-phenylalanine. Endogenous placental biotin did not contribute significantly to background staining. Despite the high level of placental alkaline phsophatase, the intestinal-type alkaline phosphatase can be used as a marker enzyme in the sensitive ABC technique, provided that the nonspecific background is measured and substracted. Moreover, it is advisable to use an acid- and/or mercuric chloride-containing fixative and to add L-phenylalanine during incubation steps.
Høyer
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PE
Institute of Medical Anatomy, Panum Institute, University of Copenhagen, Denmark.
Kirkeby
S
S
D000117:Acetylglucosamine / Q000302:isolation & purification*; D000469:Alkaline Phosphatase; D016477:Artifacts; D001360:Avidin; D016616:Cryoultramicrotomy; D006651:Histocytochemistry / Q000379:methods*; D006801:Humans; D007211:Indoles; D037102:Lectins; D016707:Tissue Fixation / Q000379:methods*; D014327:Trophoblasts / Q000166:cytology*
D007211:Indoles; D037102:Lectins; D001360:Avidin; C035455:5-bromo-4-chloro-3-indoxyl phosphate; D000469:Alkaline Phosphatase; D000117:Acetylglucosamine