CIL:40099, Rattus norvegicus, astrocyte, astrocyte of the hippocampus. In Cell Image Library

Intracellular injection of astrocytes in lightly fixed tissue slices was performed as previously described, with some modifications (Buhl et al., [1990], PMID: 2262598). The brain was placed in ice-cold PBS and cut into coronal slices with a vibratome at a thickness of 100 µm, and stored in PBS at 4°C until used. Individual cells within the slices were identified using infrared-DIC optics, impaled and iontophoretically injected with dye using 1-second pulses of negative current (0.5 Hz) for 1-2 minutes. After several cells were filled, the slices were placed in ice-cold 4% PFA for at least 1 hour. The slices were then ready to be immunolabeled. Slices were coverslipped using Gelvatol (Harlow and Lane, [1988]) and allowed to set overnight at room temperature before they were examined.
Image acquisition and analysis: Specimens were examined using a Radiance2000 laser scanning confocal system (Bio-Rad, Hercules, CA) attached to a Nikon E600FN microscope (Kanagawa, Japan). A 60x oil immersion (NA 1.4) objective was used to image filled astrocytes.