CIL:40091, 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 Alexa 568. 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 rat anti-N-CAM monoclonal antibody (isoclone 12F11) was obtained from BD PharMingen (San Diego, CA), and detected using Alexa 488. 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稍作修改，对轻度固定的脑片组织中的星形胶质细胞实施胞内注射（Buhl等，1990年，PMID: 2262598）。将脑组织置于冰预冷的磷酸盐缓冲液（Phosphate Buffered Saline, PBS）中，使用振动切片机切成厚度为100μm的冠状脑片，随后将脑片保存于4℃的PBS中待用。
将脑片置于60倍水浸物镜（数值孔径NA 1.4）下方，采用配备红外微分干涉差（Differential Interference Contrast, DIC）光学系统的Olympus BX50WI显微镜进行观察（Olympus公司，纽约州梅尔维尔市）。通过胞体的形态与尺寸，可识别齿状回上部叶片内的星形胶质细胞。
使用垂直拉针仪（David Kopf Instruments，加利福尼亚州图琼加市）拉制外径1.00 mm、内径0.58 mm、电阻为100~400 MΩ的玻璃微电极，并向电极内灌注Alexa 568染料。通过负电流脉冲（时长1秒，频率0.5 Hz）持续1~2分钟，对星形胶质细胞进行胞内穿刺并离子电渗注入染料。
待数个细胞被染料充盈后，将脑片置于冰预冷的4%多聚甲醛（Paraformaldehyde, PFA）中固定至少1小时，此时脑片即可用于免疫标记实验。大鼠抗神经细胞黏附分子（Neural Cell Adhesion Molecule, N-CAM）单克隆抗体（克隆株12F11）购自BD PharMingen公司（加利福尼亚州圣地亚哥市），并采用Alexa 488完成荧光检测。使用Gelvatol封片剂（参照Harlow与Lane，1988年的方法）对脑片进行封片，随后置于室温下静置过夜，待封片剂凝固后方可进行成像观察。
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公司，加利福尼亚州赫拉克勒斯市）对标本进行成像观察。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完成成像。