CIL:40030, 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. The slices were placed under a 60x water objective (NA 1.4) and observed with an Olympus BX50WI microscope using infrared-DIC optics (Olympus, Melville, NY). Astrocytes in the upper blade of the dentate gyrus were identified by the shape and size of their somata. Glass micropipettes (OD 1.00 mm, ID 0.58 mm; resistances 100-400 M) were pulled on a vertical puller (David Kopf Instruments, Tujunga, CA) and backfilled with either 5% aqueous LY in 200 mM KCl. Astrocytes were 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. The rabbit anti-EphA4 antibody was provided by Dr. Elena Pasquale (The Burnham Institute, La Jolla, CA). EphA4 was subsequently detected using goat anti-rabbit Cy5. 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 LY-filled astrocytes.

本研究参照既往实验方案并做部分改良（Buhl等，1990年，PMID: 2262598），对轻度固定的组织切片内的星形胶质细胞（astrocytes）实施胞内注射。将脑组织置于冰预冷的磷酸盐缓冲液（Phosphate Buffered Saline, PBS）中，经振动切片机（vibratome）切割为100 μm厚的冠状切片，随后于4℃的PBS中保存待用。将切片置于60倍水浸物镜（数值孔径NA 1.4）下方，使用配备红外微分干涉（Differential Interference Contrast, DIC）光学系统的Olympus BX50WI显微镜（Olympus，美国纽约州梅尔维尔市）进行观察。根据胞体的形态与尺寸，可识别齿状回（dentate gyrus）上翼内的星形胶质细胞。采用外径1.00 mm、内径0.58 mm的玻璃微电极（电阻范围100~400 MΩ），于垂直拉制仪（David Kopf Instruments，美国加利福尼亚州图琼加市）上拉制而成，并以200 mM氯化钾溶液配制的5%荧光黄（Lucifer Yellow, LY）水溶液对电极进行背充。通过插入细胞并施加1秒/次、频率0.5 Hz的负电流脉冲，持续1~2分钟，对星形胶质细胞进行离子电渗染料注射。待多个细胞被染色填充后，将切片置于冰预冷的4%多聚甲醛（Paraformaldehyde, PFA）中固定至少1小时，此后切片即可用于免疫标记实验。兔抗EphA4抗体由Elena Pasquale博士惠赠（美国加利福尼亚州拉霍亚市伯纳姆研究所）。后续采用山羊抗兔Cy5二抗完成EphA4的免疫检测。使用Gelvatol（Harlow和Lane，1988年）进行封片，于室温下静置过夜固化后，即可进行镜检。图像采集与分析：采用搭载于尼康E600FN显微镜（日本神奈川县）上的Radiance2000激光扫描共聚焦系统（Bio-Rad，美国加利福尼亚州赫拉克勒斯市）对标本进行观察。使用60倍油浸物镜（数值孔径NA 1.4）对LY标记的星形胶质细胞进行成像。