title: A new histochemical double-stain method using three-dimensional analysis with confocal laser scanning microscopy.
abstract: We describe a new technique for immunohistochemical and enzyme-histochemical double staining using confocal laser scanning microscopy in the reflection mode. As an example, we investigated the immunoreactivity for Spot 35-calbindin-D28K, a vitamin D-dependent calcium binding protein, and the enzyme activity for Ca(2+)-ATPase in the rat kidney. The lead precipitation method for Ca(2+)-ATPase was initially used to process kidney slices. Each specimen was then dehydrated and embedded in a water soluble resin. Thin sections were cut from the resin block, and an indirect immunocolloidal gold method with silver enhancement for Spot 35-calbindin-D28K antigen was carried out on the glass slides. Results were then observed by confocal laser scanning microscopy in the reflection mode. The three-dimensional distribution of the reaction products was detected by serial optic slice images. Lead phosphate particles, which represented the location of Ca(2+)-ATPase, were distributed deep in the section. The most intense signals from the silver particles were detected from the surface slice of the section. A stereoscopic image generated from the serial optic slices clearly showed the differences in their distribution.
authors.affiliation: Department of Medicine (II), Niigata University School of Medicine, Japan.
authors.forename: J J
authors.initials: JJ
authors.lastname: Kazama
authors.forename: T
authors.initials: T
authors.lastname: Aikata
authors.forename: M
authors.initials: M
authors.lastname: Arakawa
authors.forename: H
authors.initials: H
authors.lastname: Ozawa
mesh_terms: D000818:Animals; D064092:Calbindin 1; D064026:Calbindins; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002531:Cerebellum / Q000201:enzymology / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods*; D007668:Kidney / Q000201:enzymology / Q000378:metabolism; D007854:Lead; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D064030:S100 Calcium Binding Protein G / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: C576500:Calb1 protein, rat; D064092:Calbindin 1; D064026:Calbindins; D009419:Nerve Tissue Proteins; D064030:S100 Calcium Binding Protein G; D007854:Lead; C032722:lead sulfate; D000252:Calcium-Transporting ATPases