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

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 predictions of quantitative structure-activity relationship models of this system.

Porphycenes: Facts and Prospects in Photodynamic Therapy of Cancer

“…Likewise, some reports on the subcellular localization of porphycenes are not based on their direct visualization by fluorescence microscopy, but on the observation of PStreated cells that are also labelled with fluorescent organelle probes, i.e., a colocalization strategy is used. On the other hand, although filamentous organelles such as mitochondria are easily identified in living cells, granular components are more heterogeneous, involving a wide spectrum of ATP-dependent acidic organelles, which comprise not only endosomes and lysosomes [256,257], but also all types of zymogen granules [258,259], vacuoles [260], clathrin-coated vesicles, multivesicular bodies, and Golgi components [261].…”

Improving images of fluorescent cell labeling by background signal subtraction

“…Mechanisms for uptake and accumulation of fluorescent probes and drugs in the cytosol, endoplasmic reticulum, Golgi apparatus, lysosome, mitochondrium, and nucleus have been described (Espada et al 1997, Horobin 2001, Colston et al 2003, Horobin et al 2006, D'Souza et al 2008.…”

Fluorescence microscopy of rat embryo sections stained with haematoxylin–eosin and Masson's trichrome method

“…Minimal autofluorescence and negligible fading rate are useful features of EY, AF and XP when excited by green light. It must be emphasized that, in this case, the red colour detected by fluorescence microscopy is due to the spectral profile of the barrier filter, which allows only the far red emission of fluorescent dyes to be observed ( Espada et al ., 1997 ).…”

“…Analytical and microscopical reactions based on fluorescence emission can be at least two orders of magnitude more sensitive than those based on light absorption ( Guilbault, 1973). Therefore, there is a growing interest in converting classical staining methods for bright‐field illumination into their corresponding counterparts for fluorescence microscopy ( Ferrer et al ., 1983 ; Molero et al ., 1986 ; Raap, 1986; Espada et al ., 1993 , 1997; Stockert & Trigoso, 1994). In this connection, the main advantages of using the emission properties of EY, AF and XP (from routinely HE‐ or MT‐stained tissues) are the higher sensitivity of selective fluorescence reactions, as well as possibilities for prolonged observation, re‐evaluation of the customary histological image of archive sections, and easy recognition of specific protein structures in embryonic tissues by fluorescence microscopy.…”

“…Spectrofluorometric studies were performed with 0.1 mg mL −1 solutions of EY, XP or AF, in either distilled water or 90% glycerol, using a Perkin‐Elmer fluorescence spectrophotometer 650‐10S, equipped with a 150‐W xenon lamp and the R 372 F photomultiplier detector. To compare spectroscopic and microscopic features of the fluorescent dyes, emission spectra were also recorded through the barrier filter (BF 590) corresponding to the microscopical green excitation, which was placed between the cuvette and the detector window ( Espada et al ., 1997 ). The Raman scattering of solvents was subtracted from emission spectra.…”