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), and sections were immunostained with antibodies to the astroglial-specific cytoskeletal protein, GFAP (blue). Optical sections were generated with a single photon confocal microscope, and are shown here compiled into a maximum projection image.
Male Sprague Dawley rats, 1 week of age, were anaesthetized 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, or 20mM AlexaFluor 488 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, and subsequently immunostained. 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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