Cerium methods for light and electron microscopical histochemistry

Abstract

Summary Methods based on the use of cerium to detect the activity of oxidases and phosphatases are rapidly expanding. Both classes of enzymes can be demonstrated with cerium at the electron and light microscopical level. The in situ detection of H2O2 production with cerium is another application that has great potential, particularly in experimental pathological research. The fine precipitate of the cerium‐containing final reaction product, cerium perhydroxide or cerium phosphate, enables a very precise localization. With such techniques, important advances have been made in cell biology, such as the discovery of new organelles, functional subcompartmentization of peroxisomes, tubular lysosomes and the elucidation of the function of extracellular ATPases. At the light microscopical level, the activity of enzymes can be quantified in situ because the production of final reaction product in the cerium methods is proportional to enzyme activity in tissue sections or cells. Cerium precipitates have strong reflectance properties and this enables their use in confocal scanning laser microscopy. In the present review, the principles of cerium methods are outlined and applications in cell biology and pathology are discussed.

Localization of Hydrogen Peroxide Production in Pisum sativum L. Using Epi-Polarization Microscopy to Follow Cerium Perhydroxide Deposition

“…The little cerium deposition that occurred in CFs and vascular tissues did not appear to be much affected by the presence or absence of putrescine. One limitation to using Ce3+ on intact tissues is that these ions diffuse relatively slowly through biological samples (Van Noorden and Frederiks, 1993;Halbhuber et al, 1994); for instance, Kausch (1987) reported that they penetrate only 70 to 15 cell layers beyond the cut surface of tissue samples. Seria1 sections taken from pea epicotyl segments incubated with Ce3+ and putrescine, as well as from segments incubated in Ce3+ alone, showed fairly constant staining to depths between 100 and 200 pm (L. Liu, K.-E.L.…”

“…As far as we can determine, the only differences between the two studies were in the microscopy techniques used and the use of 1 % osmium tetroxide to enhance the Ce(IV) perhydroxide deposits for TEM observation in the study by Slocum and Furey (1991). Although postfixation with osmium tetroxide has been noted in some cases to lead to nonspecific precipitation of electron-dense materials (Okada et al, 1986;Van Noorden and Frederiks, 1993), it is difficult to see how such a phenomenon could explain the lack of staining in cortical cell walls of pea epicotyl noted by Slocum and Furey (1991). However, in the present study, the electron-dense, refractile deposits observed in cortical walls of such segments were verified to contain cerium by elemental spectroscopy (Fig.…”

“…However, these techniques are indirect measurements of H,O, production and are limited to detection of H202 produced at the cut surface of tissue sections. Briggs et al (1975) introduced the use of Ce3+ as a reagent for specific detection of H,Oz production, and this technique is now widely used for the histochemical detection of various oxidases and phosphatases (Kausch, 1987;Van Noorden and Frederiks, 1993;Halbhuber et al, 1994). Ce3+ penetrates into tissues, albeit slowly, and reacts directly with H,O, to form insoluble, finely divided deposits of Ce(1V) perhydroxide.…”

Fine Structural Specific Visualization of RNA on Ultrathin Sections

“…Data in the literature point out that these ions in their oxidation form 3 ϩ can interact with single-stranded nucleic acids (Hörer et al 1977) at their guanine sites (Ringer et al 1978), at the phosphate in unpaired residues (Topal and Fresco 1980), and especially with guanosine monophosphate in RNA (Ringer et al 1980). At the electron microscopic (EM) level, these ions (particularly lanthanum) have been known to precipitate in the presence of phosphate groups (Hayat 1993), and members of the family, such as cerium, are now routinely used for EM detection of phosphatase activity (Van Noorden and Frederiks 1993).…”

Localization of uric acid oxidase activity in core and matrix of peroxisomes as detected in unfixed cryostat sections of rat liver.

“…Only Veenhuis and Wendelaar Bonga (1979) mentioned that UAOX activity might be located in the matrix of peroxisomes. However, in more recent literature on the localization of UAOX, the protein and its activity again are described to be exclusively related to the peroxisomal core (Van Noorden and Frederiks, 1993;Volkl et al, 1988).The cerium salt capture method to demonstrate oxidase activities at the ultrastructural level can also be applied for light microscopy (LM) by using a second-step incubation in the presence of diaminobenzidine, cobalt, and Hz02 (Gossrau et al, 1989; Angermuller and Fahimi, 1988). Aqueous media can be used to demonstrate peroxisomal oxidase activities in unfixed cryostat sections (Frederiks et al, 1993b).…”