Intracellular dye injections of protoplasmic astrocytes from the CA1 region of hippocampus of a 1 week old rat reveals highly ramified spongiform processes that span territories with minimal overlap. Astrocytes were filled with Lucifer Yellow (green) and Alexa 568 (red). Optical sections were generated with a single photon confocal microscope, and compiled into the single maximum projection image shown here.
Male Sprague Dawley rats were anesthetized and perfused transcardially with with Ringer’s solution (37°C), followed by 4% paraformaldehyde (PFA) in PBS (37°C, pH 7.4). Tissue was sectioned (100 µm thick) using a vibratome, and injected with a BX50WI microscope (Olympus, Melville, NY) and, using infrared differential interference contrast videomicroscopy,cells with a small diameter soma (approx. 7–11µm) in the stratum radiatum of CA1 were located and impaled with glass micropipettes filled with either 5% aq. LY, 20mM AlexaFluor 568 in 200mM KCl. The astrocytes were filled using a 0.5 Hz pulses of current until all of the processes were brightly fluorescent. After several astrocytes were filled, a slice was placed in the 4% PFA overnight at 4°C. Slices were then imaged after coverslipping in Gelvatol. Confocal images were acquired with a BioRad Radiance2000 (Hercules, CA) attached to a Nikon E600FN (Kanagawa, Japan) microscope equipped with a Nikon (Tokyo, Japan) 60× oil objective (NA 1.4). Additional details are provided in Bushong EA, Martone ME, Ellisman MH. Maturation of astrocyte morphology and the establishment of astrocyte domains during postnatal hippocampal development. Int J Dev Neurosci. 2004 Apr;22(2):73-86.
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