培养细胞透射电镜样品冷冻制备技术

冷冻制备的生物样品最适合于免疫细胞化学的研究．本文用３Ｔ３－Ｌ１培养细胞经多聚甲醛和戊二醛混合液对样品进行短暂的固定，加明胶液置微离心管，制成细胞团块，４℃冷凝，经后固定，将细胞团修成＜１ｍｍ３的块头，密封于蔗糖中，用液氮冷冻．其优点是：１）经多聚甲醛戊二醛混合液的短暂固定，不但保持良好的免疫组化染色效果，且冷冻样品更适合于形态观察；２）用明胶液及微离心管离心即使较少量的细胞数也可获得理想的细胞团块；３）第２次固定使细胞团块不易松散；４）其冷冻包埋块可同时做冷冻半薄切片和超薄切片分别供光镜和电镜观察研究

Preparation of Cultured Cells for Cryosectioning in Electronmicroscopic Level

The biological sample preparation for cryosectioning in electronmicroscopic level is a useful technique for immunolabeling.In this study,cultured 3T3-L1 cells were first fixed by 3%(w/v) parafornadehyde and 0.2% glutaraldehyde,spun in a micro centrifuge tube to form the cell pellet. The cell pellet was cut with the tube and fixed again,then the blocks (<1 mm 3) were made and cryo embedded in liquid nitrogen with sucrose. The advantages of this procdure are. 1) Fixation with both parafornadehyde and glutaraldehyde protects the samples from damaging and withdrawing the macromolecules by the fixing reagents .2) Using micro centrifuge tube is a easy way to get perfect cell pellet,especially when the amount of cells are not abundant enough.3) The second fixation makes the cell pellet more solid for further handling.4) The procedure provides blocks suitably for simi thin and ultrathin cryosectioning for immunolabeling in both light and electronmicroscopic levels.