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Fig. 7. Two-by-two combinations of anti D-titin antibody staining in metaphase insect spermatocytes. (A) One confocal slice showing the distribution of anti-56 antibody (green) and anti-52 antibody (red) staining in metaphase locust spermatocytes. The two antibodies show a punctate distribution of D-titin, which is localized in each case along the kinetochore spindle fibers (merged image) with little co-localization (yellow-orange color). (B) Enlarged region of the boxed region marked in A, showing the regular, punctate distribution of anti-56 antibody (green) and anti-52 antibody (red) staining and the co-localization of different D-titin fragments along one kinetochore spindle fiber. (C) One confocal slice showing the distribution of anti-KZ antibody (green) and anti-52 antibody (red) staining in metaphase crane-fly spermatocytes. The two antibodies show the same pattern of punctate distribution as in A and localize along the kinetochore spindle fibers (merged image), with little co-localization. (D) Enlarged details of the boxed region in C, showing the regular, punctate distribution of anti-KZ antibody (green) and anti-52 antibody (red) staining and the co-localization (yellow-orange) along one kinetochore spindle fiber. Three consecutives slices illustrate their spatial localization in the Z-axis. (E) Distribution of anti-56 antibody (green) vs anti-KZ antibody (red) in a metaphase crane-fly spermatocyte. The two antibodies show the same pattern of punctate distribution as in A and C and localize along the kinetochore spindle fibers (merged image), with little co-localization except in the chromosomes (arrowheads) and at the poles (open arrowheads). The arrows indicate a flagellum extending from a spindle pole. (F) RNase treatment of crane-fly spermatocytes during the staining protocol resulted in increased chromosomal staining with anti-KZ antibody, thus masking the spindle staining (left panel) whereas anti-56 antibody staining remained unchanged, both in its localization and intensity (right panel) (cf. Fig. 4A, untreated). Both panels depict the same cell. Arrowhead indicates a sex chromosome and arrow indicates an autosomal bivalent. Bars, 5 µm.
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