A New Histochemical Double-Stain Method Using Three-Dimensional Analysis with Confocal laser Scanning Microscopy
作者:
KazamaJunichiro J.,
AikataTakashi,
ArakawaMasaaki,
OzawaHidehiro,
期刊:
Biotechnic&Histochemistry
(Taylor Available online 1994)
卷期:
Volume 69,
issue 6
页码: 324-328
ISSN:1052-0295
年代: 1994
DOI:10.3109/10520299409106313
出版商: Taylor&Francis
关键词: confocal laser scanning microscopy (CLSM);reflection mode;three-dimensional image;calcium-ATPase;Spot 35-Calbindin-D28K;double staining
数据来源: Taylor
摘要:
We describe a new technique for immunohistochemical and enzyme-histochemi-cal 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 ma+-ATPase in the rat kidney. The lead precipitation method for Ca2+-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-D28Kantigen 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 Ca2+-ATPase, were distributed deep in the section. The most intense signals from the silver partkles 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.
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