PMID: 9450630Feb 5, 1998Paper

Fluorescent cytochemistry of acid phosphatase and demonstration of fluid-phase endocytosis using an azo dye method

Histochemistry and Cell Biology
J EspadaJ C Stockert

Abstract

The aim of this work was the development of a fluorescent microscopy technique to visualize acid phosphatase activity in living and pre-fixed cells. We have shown that a coupling azo dye method, using naphthol AS-MX phosphate (NP) as substrate and fast red TR (FR) as a diazonium salt coupling agent, gives rise to a fluorescent azo dye reaction product which permits a highly sensitive demonstration of lysosomal acid phosphatase in both living and pre-fixed monolayer cell cultures. The granular staining is prevented by inhibition of acid phosphatase activity using fluoride and/or orthovanadate in both living and pre-fixed preparations. Lysosomal staining in living cells is also abolished by inhibition of fluid-phase endocytosis using low temperatures or inhibition of oxidative phosphorylation. It was shown that whilst NP entered living cells by passive diffusion, occurrence of FR in lysosomes resulted from fluid-phase endocytosis. Spectroscopic analysis of the emission and absorption features of FR, NP, naphthol AS-MX (N), and the N-FR azo dye reaction product in solution corroborated our microscopic results. The differing uptake mechanisms, and the occurrence of lysosomally localized azo dye, were also in keeping with the predic...Continue Reading

Citations

Apr 30, 2005·Analytical Chemistry·Chandra M DasPeter R C Gascoyne
Mar 10, 2009·Biotechnic & Histochemistry : Official Publication of the Biological Stain Commission·J C StockertM Cañete
Feb 25, 2003·Annals of Anatomy = Anatomischer Anzeiger : Official Organ of the Anatomische Gesellschaft·Karl-Jürgen Halbhuber, Karsten König

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