Tsuyama Shinichiro, Matsushita Sachie, Takatsuka Tomio, Nonaka Satoru, Hasui Kazuhisa, Murata Fusayoshi
Department of Anatomy, Kagoshima University, Faculty of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan.
Anat Sci Int. 2002 Mar;77(1):74-83. doi: 10.1046/j.0022-7722.2002.00011.x.
Gastric gland component cells were electron-microscopically and immunoelectronmicroscopically examined with high-pressure freezing followed by freeze substitution and a low-temperature embedding resin method and compared to that of the conventional chemical-fixation method. The rat gastric gland was high-pressure frozen, freeze-substituted with acetone-containing osmium or acrolein, and embedded in Epon 812 or Lowicryl K4M, respectively. Using the high-pressure freezing method, the vitreous freezing range reached the depth of 150 microns from the surface. The ultrathin sections from both procedures embedding in Epon 812 and Lowicryl K4M were doubly stained with uranyl acetate and lead acetate, and histochemically or immunohistochemically stained, respectively. In comparison to the conventional chemical fixation method, excellent results were obtained with respect to ultrastructural preservation. The stainings performed in this experiment included periodic acid-thiocarbohydrazide-silver proteinate staining, cationic colloidal cold at pH 2.5 staining, Helix pomatia lectin-staining, anti-alpha or -beta subunit antibodies of H+K(+)-ATPase immunostaining and pepsinogen immunostaining. The staining intensity of those was stronger than that of the conventional immersion-chemical fixation method. In addition to these results, the labels also showed good specific localization. In this paper, we provide a description of the high-pressure freezing followed by freeze substitution and low-temperature embedding resin method compared to the conventional chemical-fixation method. Our results suggest that this method is a suitable tool for ultrastructural and histochemical/immunohistochemical studies at high resolution.
采用高压冷冻、冷冻置换及低温包埋树脂法，对胃腺组成细胞进行电子显微镜和免疫电子显微镜检查，并与传统化学固定法进行比较。将大鼠胃腺进行高压冷冻，用含丙酮的锇或丙烯醛进行冷冻置换，然后分别包埋于Epon 812或Lowicryl K4M中。采用高压冷冻法，玻璃化冷冻范围可达距表面150微米的深度。将包埋于Epon 812和Lowicryl K4M的两种方法制备的超薄切片分别用醋酸铀和醋酸铅进行双重染色，并分别进行组织化学或免疫组织化学染色。与传统化学固定法相比，在超微结构保存方面取得了优异的结果。本实验进行的染色包括高碘酸-硫代碳酰肼-银蛋白染色、pH 2.5时的阳离子胶体冷染色、苹果蜗牛凝集素染色、H⁺K⁺-ATP酶α或β亚基抗体免疫染色和胃蛋白酶原免疫染色。这些染色的强度均强于传统的浸入式化学固定法。除了这些结果外，标记物还显示出良好的特异性定位。在本文中，我们描述了与传统化学固定法相比的高压冷冻、冷冻置换及低温包埋树脂法。我们的结果表明，该方法是一种适用于高分辨率超微结构和组织化学/免疫组织化学研究的工具。
