包埋前原位TUNEL标记凋亡淋巴细胞的电镜观察

Electron Microscopic Observation of Apoptosis in Pre-Embedding TUNEL-Labeled Lymphocytes in Mouse Lymph Nodes

目的用包埋前原位尾端标记技术在电子显微镜下发现小鼠淋巴结生发中心早期凋亡细胞。方法用GA,PA,PLP分别固定淋巴组织,将其分别切成50μm切片,TUNEL染色,制成1μm切片光镜确认,着色部位制成超薄切片,在电镜下,进行比较观察。结果GA固定的组织中细胞核的TUNEL染色,虽然表面清晰可见,但对组织渗透性较差;PA固定的组织清晰度稍差,但渗透性最好,在电子显微镜下观察效果满意,PLP固定染色效果差,在细胞凋亡的早期,用PA染色时凋亡的细胞核内,可见尚未出现凋亡的生发中心细胞核形态学改变以及核染色质浓缩的核。结论以PA固定的组织,用包埋前技术、TUNEL染色的方法具有简便,染色清晰,易分辨,特异性强的特点,且未见标本损坏现象。

Objective To study the early apeptotic germinal center cells in mouse lymph nodes pre-embedding TUNEL- labeled and observed by transmission electron microscopy. Methods The tissue of mouse lymph nodes were cut into 50μtm and fixed in glutaraldehyde （GA）, paraformaldehyde/PBS （PA） or periodate-lysine-2 % paraformaldehyde （PLP）, labeled by TUNEL, and 1 μm sections were examined under the optical microscope. The uhrathin sections were observed by transmission electron microscopy. Results The GA-fixed and TUNEL-labeled tissues specimens were clearest, hut the fixative penetration was weak. The PA-fixed tissues specimens were better, with best fixative penetration. The PLP-fixed tissues failed to he labeled by TUNEL. In the early stage of apeptosis, the nuclei in PA-fixed lymphocytes were well stained, depicting clear and well stained nuclear chromation and condensed nucleolus. Conclusion In PA-fixed tissue, pre-embedding TUNEL labeling is a simple and reliable technique to demonstrate good morphology of early apeptotic alterations with high specificity, without distinct tissue damages.