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 (green emission) and Alexa Fluor 568 (red emission), respectively. Cell nuclei were subsequently labeled with a far-red fluorescent DNA probe, DRAQ5 (pseudocolored blue). Images were recorded with a 20x objective using a zoom factor of 1.5 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.
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