Reflectance enzyme histochemistry (REH): visualization of cerium-based and DAB primary reaction products of phosphatases and oxidases in cryostat sections by confocal laser scanning microscopy

Abstract

In the present study the reflectance mode of confocal laser scanning microscopy was adapted to detect and to assess semiquantitatively cerium-based primary reaction products of oxidases [Ce(IV) perhydroxide] and phosphatases [Ce(III) hydroxyphosphate converted into Ce(IV) perhydroxyphosphate] as well as of the 3,3'-diaminobenzidine (DAB)-based primary reaction product of cytochrome c oxidase in cryostat sections. Confocal laser scanning microscopy offers a unique way of making visible histochemical reaction products which are weakly absorbant but sufficiently reflective. It was easily possible to record simultaneously the reflectance signals at the wavelength of the exciting laser (preferentially 488 nm) and the autofluorescence signals ( > 580 nm in our set-up) of glutaraldehyde-fixed tissue. The results of an imbibition study of cerium-containing model precipitates indicate that the cerium, generally, should be oxidized prior to observation because the index of refraction of Ce(IV) compounds is considerably higher than that of the corresponding Ce(III) compounds. An attempt at comparative numerical assessment of reflection intensities from reflectant parts in morphologically similar sections is presented. The proposed technique may open new possibilities in enzyme- and immunohistochemistry.

Low dose (−)deprenyl is cytoprotective: It maintains mitochondrial membrane potential and eliminates oxygen radicals

“…The present cerium staining used for the in situ detection of intracellular ROS is the adaptation of previous methods used by Robinson and Batten (1990), Van Norden and Frederiks (1993), Halbhuber et al (1996), Bestwick et al (1997) and Telek et al (1999Telek et al ( , 2001. Its limitation, however, similarly to all the available assays, that it is not rigorously quantitative, since the dye competes with other molecules for the interaction with ROS.…”

“…Fluorescence present in the cells was measured at 488-nm excitation/ 510-to 625-nm emission. The reaction of cerium ions with ROS forms stable, insoluble cerium perhydroxide (Ce III [OH] 2 OOH or Ce IV [OH] 3 OOH precipitates detectable with reflectance methods (Robinson and Batten, 1990;Van Norden and Frederiks, 1993;Halbhuber et al, 1996;Bestwick et al, 1997;Telek et al, 1999Telek et al, , 2001.…”

A Technical Triade for Proteomic Identification and Characterization of Cancer Biomarkers

“…In most cases, Zeiss objective NEOFLUAR 40ϫ/1.30 oil was used in combination with pinhole 20 at a scanning time of 60 s (23,24).…”

Two‐photon microscopy of deep intravital tissues and its merits in clinical research

“…Under CLSM microscope, not only the in-focus but also the out-of-focus molecules can be excited (Halbhuber et al, 1998). The emitted fluorescence signals of the both areas are collected.…”