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Journal of Histochemistry and Cytochemistry, Vol. 47, 1343-1349, October 1999, Copyright © 1999, The Histochemical Society, Inc.

TECHNICAL NOTE

Quantification of Protein A–Gold Staining for Peroxisomal Enzymes by Confocal Laser Scanning Microscopy

Naoko Ogiwaraa, Nobuteru Usudaa, Masashi Yamadac, Kohei Johkuraa, Kiyokazu Kametanib, and Ayami Nakazawaa
a Department of Anatomy and Cell Biology, Shinshu University School of Medicine, Matsumoto, Japan
b General Research Laboratory, Shinshu University School of Medicine, Matsumoto, Japan
c Leica K.K., Tokyo, Japan

Correspondence to: Nobuteru Usuda, Dept. of Anatomy and Cell Biology, Shinshu Univ. School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan.

The protein A–gold technique has been widely applied for visual localization and quantification of various antigens by electron microscopy. Observation of specimens stained by the protein A–gold technique with conventional light microscopy is difficult because of insufficient sensitivity of the staining. Light microscopic visualization and quantification of the reaction products were attempted employing a confocal laser scanning microscope (CLSM). Liver tissues of normal and peroxisome proliferator-treated rats were fixed and embedded in Lowicryl K4M resin. Ultrathin and thin sections were stained for catalase and a peroxisome-specific ß-oxidation enzyme by the protein A–gold technique. Ultrathin sections were observed by electron microscopy and the labeling density for each enzyme was analyzed with an image analyzer. Thin sections were observed with a CLSM in the reflection mode and the intensity of the light reflection was analyzed under the same conditions for all specimens. A comparison of these two observation procedures was also attempted using liver tissues stained with various concentrations of the antibody for catalase. The intensity of the reflection for each, as observed by CLSM, correlated well with the labeling density observed by electron microscopy. CLSM made it possible to quantify and to directly observe protein A–gold staining at the light microscopic level. (J Histochem Cytochem 47:1343–1349, 1999)

Key Words: confocal laser scanning, microscopy, protein A–gold technique, peroxisome, liver, quantification


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