Dynamics of Carbohydrate Affinities at the Cell Surface: MATERIALS AND METHODS(13)

Replicate experiments (n = 3) were performed on separate days using fresh sperm from two bulls.

Toluidine Blue Staining of the Oviducts

Oviducts were obtained from a local slaughterhouse (Gosschalk and Zn BV) and the oviduct connected to the ovary with the dominant follicle was used. The isthmus and ampulla were dissected into 50-mm pieces and placed into Bouin fixative for 24 h. After fixation, tissue was dehydrated, embedded in paraffin, and cut into 10-^m sections. Sections were mounted on poly-L-lysine-coated slides, deparaffinized in xylene, and rehydrated in graded ethanol dilutions (100%, 90%, 80%, 70%, and 50%). Rehydrated sections were stained with toluidine blue (0.1% [w/v] in phosphate buffer, pH 5.5) for 5 min.  After rinsing in water, slides were dehydrated in a graded series of ethanol (70%, 80%, 90%, and 100%) and in xylene.Slides were mounted with DePeX mounting medium and analyzed using bright-field illumination under a BH-2 microscope (Olympus). The resulting toluidine blue-stained histological slides of isthmus and ampulla of cow oviducts were used to detect the distribution of sulfated GAG (pink staining) on a blue background staining for other cellular material.

Statistics

For statistical analysis, the data were fitted into a one-site binding curve (i.e., E450 = Bmax X [fucose-PAA-biotin]/(Kd + [fucose-PAA-bio-tin]) where Bmax is the maximal binding and Kd is the concentration of fucose-PAA-biotin required to reach half-maximal binding). The individual curves were compared with a curve of the combined data using F-statistics, in which the sum of squares of the individual curves were compared with the sum of squares of the curve of the combined data set. Statistical significance was considered when P < 0.05.

Unconjugated carbohydrates were tested for binding competition to biotinylated carbohydrates and 50% binding inhibition (EC50) was determined using a one-site competition curve (E450 = minE450 + (maxE450 -minE450)/(1 + 10([carbohydrate]-log(EC50))). The software used was GraphPad Prism (GraphPad Software, Inc., San Diego, CA).

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