细胞样本透射电镜实验步骤

一、实验器材及试剂

1、 实验器材

2、 主要实验试剂

二、透射电镜制片步骤

1、取材固定：离心收集细胞或细菌沉淀，4°C固定保存及运输。

2、琼脂预包埋：细胞悬液用离心机1000-2000rpm离心5-10min(根据实际情况适当增加速度)，弃上清加入 0.1M 磷酸缓冲液 PB（PH7.4），混匀漂洗 5min 后再1000-2000rpm离心5-10min(根据实际情况适当增加速度)，重复洗涤 3 次。弃去上清，细胞沉淀中提前加热溶解制备 1%琼脂糖溶液，稍冷却后加入 EP 管内，在琼脂糖凝固之前将细胞沉淀用牙签挑起悬浮包裹于琼脂糖内，细胞丰富区域切成1mm³大小。

3、后固定：0.1M 磷酸缓冲液 PB（PH7.4）配制的 1%锇酸避光室温固定 2h。0.1M 磷酸缓冲液 PB（PH7.4）漂洗 3 次，每次 15min。

4、 室温脱水：组织依次入 30%-50%-70%-80%-95%-100%-100%酒精上行脱水每次 10min，

100%丙酮两次，每次 10min。

5、 渗透包埋：丙酮︰812 包埋剂=1︰1， 37℃ 2-4h， 丙酮︰812 包埋剂=1︰2 ，37℃渗透过夜， 纯 812 包埋剂 37℃ 5-8h。将纯 812 包埋剂倒入包埋板，将样品插入包埋板后 37℃烤箱过夜。

6、 聚合：包埋板放于 60℃烤箱聚合 48h，取出树脂块备用。

7、 超薄切片：树脂块于超薄切片机 70nm 超薄切片，200 目方华膜铜网捞片。

8、 染色：铜网于 2%醋酸铀饱和酒精溶液避光染色 10min；超纯水清洗3 次；枸橼酸铅溶液避二氧化碳染色 5-10min；超纯水清洗 3 次，滤纸稍吸干。铜网切片放入铜网盒内室温干燥过夜。

9、透射电子显微镜下观察

  TEM staining report for cells and bacteria

1     Apparatus and reagents

1.1 Major apparatus

2     Procedure

2. 1  Harvest   samples  and  fixation:  Collect  cells  or  bacteria  precipitation   after  centrifuge. The TEM fixative was added to the tube and let the precipitation re-suspended in the fixative, and then fixed at 4℃ for preservation and transportation.

2.2 Agarose pre-embedding: The fixed cells and bacteria were centrifuged. The 0. 1 M PB (pH 7.4) was  added  into the tube  after  supernatant was  discarded,  and  then the precipitation  was re-suspended and washed in PB for 3min. This washing step was repeated for 3 times. The  1% agarose  solution  was prepared by heating  and  dissolving  in  advance. After  being  cooled,  the agarose solution was added into the EP tube. Before agarose solidification, the precipitation was suspended with toothpick and wrapped in the agarose.The size of cells   precipitation should be no more than 1 mm3

2.3 Post-fix: Agarose blocks with samples avoid light post fixed with  1% OsO4  in 0. 1 M PB (pH 7.4) for 2 h at room temperature. After remove OsO4, the tissues are rinsed in 0. 1 M PB (pH 7.4) for 3 times, 15 min each.

2.1  Dehydrate at room temperature as followed:

30% ethanol for 10 min;

50% ethanol for 10min; 70% ethanol for 10min; 80% ethanol for 10min; 95% ethanol for 10min;

Two changes of 100% ethanol for 10 min;  Finally two changes of acetone for 10min.

2.4 Resin penetration and embedding as followed:

Acetone：EMBed 812= 1:1for 2-4h at37℃

Acetone：EMBed 812= 1:2 overnight at 37℃

pure EMBed 812 for 5-8 h at 37℃;

Pour the pure EMBed 812 into the embedding models and insert the tissues into the pure EMBed 812, and then keep in 37℃ oven overnight.

2.5 Polymerization: The embedding models with resin and samples were moved into 60℃ oven to polymerize for more than 48h. And then the resin blocks were taken out from the embedding models for standby application at room temperature.

2.6 Ultrathin section: The resin blocks were cut to 60-80nm thin on the ultra microtome, and the tissues were fished out onto the 200 meshes cuprum grids with formvar film.

2.7 Staining: 2% uranium acetate saturated alcohol solution avoid light staining for 10 min,  then rinsed in ultra pure water for 3 times.  Lead citrate avoid CO2  staining for 10 min, and then rinsed with ultra pure water for 3 times. After dried by the filer paper, the cuprum grids were put into the grids board and dried overnight at room temperature.

2.8  Observation  and  images  capture:  The  cuprum  grids  are  observed  under TEM  and take images.