Double-label confocal laser-scanning microscopy, image restoration, and real-time three-dimensional reconstruction to study axons in the central nervous system and their contacts with target neurons.

The current double tracing-double confocal laser-scanning method was developed to reconstruct identified nerve fibers and their contacts with identified target neurons in the rat brain in three dimensions. It intends to fill the gap between conventional light microscopic and electron microscopic neuroanatomic tracing. The steps involved are as follows: (1) injection of two neuroanatomic tracers--Phaseolus vulgaris leucoagglutinin (PHA-L) to label fibers innervating a particular brain area and Neurobiotin to label prospective target neurons in that area; (2) immunofluorescence detection of the labeled fibers (fluorophore Cy5, infrared emission), together with fluorochromated avidin detection of the taken-up Neurobiotin (Cy2 or Alexa 488; green emission); (3) acquisition of Z-series of confocal images at high magnification with a laser-scanning microscope using the laser lines 488 nm and 647 nm; and (4) computer-processing and three-dimensional reconstruction of the labeled fibers and the presumed target dendrites.

Two-laser dual-immunofluorescence confocal laser scanning microscopy using Cy2- and Cy5-conjugated secondary antibodies: unequivocal detection of co-localization of neuronal markers.

The ability of the confocal laser scanning microscope (CLSM) to visualize in one focal plane the fluorescence associated with multiple markers renders this instrument extremely valuable for the study of co-localization of various markers in the somata and cellular processes of neurons. In the present protocol we deal with the question whether or not co-localization exists in neurons of two different neuronal markers. The conventionally used method towards answering this type of question is double-immunofluorescence microscopy.

Phagocytosis and killing of suspended and adhered bacteria by peritoneal cells after dialysis.

Objective: To determine the effect of dialysis fluid containing various glucose concentrations on the phagocytosis and killing of Staphylococcus aureus by rat peritoneal cells under conditions mimicking the in vivo situation.

Design: Phagocytosis and killing were evaluated by quantitation of the killing capacity of macrophages after in vivo phagocytosis of the bacteria as well as by an in vitro flow cytometric assay of the phagocytosis and killing of adhered bacteria by peritoneal cells.

Animals: Male Wistar rats.

Novel isolation and purification method permitting functional cytotoxicity studies of macrophages from milky spots in the greater omentum.

Milky spots in the greater omentum are well organized perivascular infiltrates of leukocytes which are probably involved in the clearance of tumor cells from the peritoneal cavity. In milky spots, macrophages are the predominant cell type forming a distinct population of cells. To investigate whether these macrophages have a function in the control of metastatic spread in the peritoneal cavity, a novel isolation and purification method was developed in order to study the functional cytotoxicity of macrophages from milky spots in the greater omentum against tumor cells in vitro.

Separation of alveolar macrophages and dendritic cells via autofluorescence: phenotypical and functional characterization.

In a previous study we demonstrated that an increase of monocytes and dendritic cells (MDC) was In the current study, the bright autofluorescence of alveolar macrophages (AMs) was used to separate them efficiently from the MDC. Sorting of freshly isolated BAL cells resulted in a high-autofluorescent fraction, consisting predominantly of AMs, and a low-autofluorescent fraction containing the MDC, lymphocytes, and granulocytes. Thus, a clear separation between suppressive (AM) and stimulating (MDC) activity was obtained as shown in antigen-specific T cell responses.