Confocal Microscopy Image Gallery
Rat Brain Tissue Sections
The hippocampus is closely associated with two other structures of the brain, the dentate gyrus and the subiculum. Together the structures comprise an assembly known as the hippocampal formation that in humans lies deep within the parahippocampal gyrus.
Considered part of the limbic system, the hippocampal formation plays an important role in memory, learning, and the realization of novelty. The hippocampus was also considered according to early theories of emotion regulation to function in the coordination of input from the cerebral cortex and the hypothalamus. However, later research with primate subjects indicated that the coordination that was thought to be performed by the hippocampus is actually carried out by another part of the limbic system, the amygdala. The hippocampus is now considered to only indirectly affect emotional states.
The hippocampus and other components of the hippocampal formation are morphologically simpler than the neocortex that overlies them. The structures exhibit a laminar organization, but unlike the neocortex, are each composed of three primary cell layers rather than six. Considered phylogenetically older than the neocortex, the cortical material that comprises the hippocampal formation is referred to as archicortex. The three strata of the hippocampus and the subiculum are the molecular, pyramidal, and polymorphic layers. The third division of the hippocampal formation, the dentate gyrus, is similarly organized, but features a granule cell layer rather than a pyramidal layer.
The coronal section of rat hippocampus tissue presented above was immunofluorescently labeled for heavy chain neurofilament subunits with chicken anti-NF-H antibodies followed by goat anti-chicken secondary antibodies conjugated to Alexa Fluor 568 (yielding red fluorescence). Neurofilaments are specialized intermediate filaments that are most prevalent in the axons of neurons, but are also present in dendrites and the neuronal cell body. In addition, the calcium-binding protein calbindin was targeted in the brain section with mouse anti-calbindin antibodies visualized with goat anti-mouse secondary antibodies conjugated to Alexa Fluor 488 (green fluorescence). Cell nuclei were counterstained with DRAQ5 (pseudocolored blue), a red-absorbing fluorescent probe. Images were recorded with a 20x objective using a zoom factor of 1.7 and sequential scanning with the 488-nanometer spectral line of an argon-ion laser, the 543-nanometer line from a green helium-neon laser, and the 633-nanometer line of a red helium-neon laser. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles unless otherwise noted above.
Additional Confocal Images of Rat Hippocampus Tissue Sections
Distribution of beta-Tubulin and Neurofilaments in a Rat Hippocampus Coronal Section - In a double immunofluorescence experiment, a coronal rat hippocampus tissue section was labeled for the neuron-specific class III beta-tubulin isoform and heavy chain neurofilament subunits by treating the specimen with a cocktail of mouse anti-beta-III-tubulin and chicken anti-NF-H primary antibodies followed by goat anti-mouse and anti-chicken secondary antibodies (IgG) conjugated to Alexa Fluor 488 and Alexa Fluor 568, respectively. Nuclear DNA was counterstained with DRAQ5.
Employing Immunofluorescence to Label Rat Brain Tissue for Calbindin and NF-H - Calbindin is a calcium-binding protein belonging to the troponin C superfamily expressed in the mammalian brain, kidney, and pancreatic islets. This protein and heavy chain neurofilament subunits were simultaneously targeted in this rat hippocampus section (coronal) with mouse anti-calbindin and chicken anti-NF-H monoclonal antibodies. The primary targets were visualized with goat anti-mouse and anti-chicken secondaries conjugated to Alexa Fluor 488 and Alexa Fluor 568, respectively. Cell nuclei were subsequently labeled with a far-red fluorescent DNA probe, DRAQ5.
Nathan S. Claxton, Shannon H. Neaves, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.