Immunocytochemical localization of the vitamin D-induced calcium-binding protein: relocation of antigen during frozen...

The vitamin D-induced calcium-binding protein (CaBP) was localized in chick duodenum by the indirect fluorescent antibody technique after tissue was prepared by three different rapid-freezing methods: freeze-thaw, freeze-drying, and freeze-substitution. Sections prepared by freeze-thawing demonstrated CaBP-specific fluorescence over goblet cells and at the absorptive surface of villi, but not in absorptive cells. Sections from the same or adjacent segments prepared by freeze-drying or freeze-substitution produced virtually the opposite pattern of CaBP distribution. CaBP-specific fluorescence was associated with absorptive cell cytoplasm but not with goblet cells. From the results of experiments in which 6 micrometer freeze-dried sections were rehydrated, it was concluded that in the presence of an aqueous environment CaBP migrated from absorptive cells to discrete localization sites bound to goblet cell mucus, possibly to the calcium present in the mucus. By analogy, it was concluded that CaBP in association with goblet cells and the absorptive surface in sections prepared by the freeze-thaw method represented an artifactual localization. The true in situ localization of CaBP in chick duodenum was that which was present in absorptive cell cytoplasm.