【原创】广州什么地方可以做冰激淋透射电镜？

2.5电镜样品准备
用于TEM分析的冰激淋样品（大约100 mm3）在-25℃下取样，用外科手术柳叶刀将样品放入-196℃液氮中，冰激淋变成＜1.0 mm3大小的颗粒。将冰冻的颗粒转移到含有3%(v/v)的戊二醛无水甲醇溶液中，在-80℃下放置4天。再将样品在-40℃下放置1天，然后转移到-20℃下放置2天。先用-20℃预冷的无水甲醇洗固定好的样品，然后用-20℃预冷的无水乙醇洗。然后依次用体积比3：1的甲醇/ LR-金色树脂（LR-gold resin），1：1的甲醇/LR-金色树脂和1：3的甲醇/ LR-金色树脂洗，最后用100%的LR-金色树脂洗。然后将样品放入含0.1%(w/v)二苯（基）乙二酮(benzyl)的LR-金色树脂的胶囊里过夜。LR-金色树脂在-20℃ 360nm UV灯下浸透到样品里面发生聚合。将树脂块用LKB超微切片机（Leica Reichert Ultracut S,Vienna,Austria）切成90nm的薄片并立即放入用镍涂布的小格栏(Marivac Ltd., Halifax, NS, Canada)里。
2.6染色和电镜实验
采用Hillbrick,McMahon和McManus(1999)报道的方法进行免疫标记。阻滞剂为2%(w/w)牛血清白蛋白的20mM磷酸盐缓冲液（PBS,pH 7.4）。含有超薄切片的格栏用数滴阻滞剂漂洗20min以防止非特异性的抗体结合。用磷酸缓冲液将一抗（primary antibody）稀释200倍，在潮湿箱内将格栏内的超薄切片漂洗1h。空白用不含一抗的PBS漂洗。格栏用PBS洗4次接着用数滴PBS再洗（四次，每次2min）。为了确定一抗的位置，格栏再用稀释20倍（PBS稀释）的二抗(secondary antibodies)在室温下的潮湿箱内漂洗2h。格栏用PBS洗4次接着用数滴PBS再洗(洗4次，每次2分钟)，最后用数滴蒸馏水洗（洗4次每次2min）。格栏然后用2%(w/w)乙酸双氧铀(uranyl acetate)溶液染色10min以强化对比度。TME用LEO-912AB显微镜（Munster,Germany）在100kV下操作。图片用Proscan CCE摄像机和SIS Esivision软件公司的SIV（图像观看软件）观看。每个冰激淋样品至少做成三个树脂样品，每个树脂样品至少检测三个薄片而且每个薄片至少检测20个不同的区域。

英文原文
2.5. Sample preparation for transmission electron
microscopy (TEM)
Ice cream specimens (approximately 100mm3) for TEM were taken from the inner bulk of the hardened samples at-25℃with a surgical blade and immediately placed into liquid nitrogen (-196℃), where they were broken into o1mm3 pieces. The freeze-substitution technique was based on that of Goff et al. (1999).Frozen specimens of ice cream were transferred into vials that contained 3% (v/v) glutaraldehyde in absolute methanol, which had been kept at -80℃ for 4 days. Although minimal fixation is important for immunogold labeling, to prevent loss of antigenicity, the use of glutaraldehyde was possible due to the enhanced
sensitivity that arises from the use of polyclonal antibodies compared to monoclonal antibodies. Samples were transferred t-40℃ for 1 day, during which the fixative mixture melted, and gradual freeze substitution of ice with methanol and some fixation with
glutaraldehyde took place. Samples were then transferred to-20℃ for 2 days where fixation proceeded at a faster rate. The fixative mixture was replaced by washing the specimens in precooled (-20℃) 100% methanol followed by washing with absolute ethanol. Infiltration of specimens with resin was carried out by graded washing with a mixture of methanol and LR-gold resin at volume ratios of 3:1, 1:1 and 1:3, followed by washing in 100% resin. Specimens were placed into gelatin capsules filled with resin containing 0.1% (w/v) benzyl and held overnight. Then the resin-infiltrated samples were polymerized under 360 nm UV light at -20℃. Resin blocks were sectioned at a thickness of 90nm using an LKB ultramicrotome (Leica Reichert Ultracut S, Vienna, Austria). Sections were immediately mounted on nickel-coated grids (Marivac Ltd., Halifax, NS, Canada).
2.6. Immuno-gold labeling and TEM
Immunolabeling was based on the methods described by Hillbrick, McMahon, and McManus (1999). The blocking agent was comprised of 2% (w/w) bovine serum albumin in 20mm phosphate buffer saline (PBS, pH 7.4). Grids containing the sections were floated for 20 min on drops of blocking agent to prevent nonspecific binding of the antibodies. The grids were floated in a humidity chamber on the primary antibody diluted 1:200 with PBS for 1 h. The control was floated on PBS instead of the primary antibody. Grids were rinsed four times with PBS followed by floating on drops of PBS (four times at 2 min each). To localize the positions of the primary antibodies the grids were then floated on the secondary antibodies diluted 1:20 with PBS for 2 h in a humidity chamber at room temperature. Grids were rinsed with PBS (four times) followed by floating on drops of PBS (four times at 2 min each), and were finally washed by floating on drops of distilled water (four times at 2 min each). The grids were then stained with 2% (w/w) uranyl acetate solution for 10 min to enhance the contrast. TEM was performed with a LEO-912AB microscope (Munster, Germany) operated at 100 kV. The images were examined using a Proscan CCE camera and SIV (soft imaging viewer) from SIS Esivision software. At least three blocks of each ice cream sample were sectioned, and at least three sections per block and 20 fields per section were viewed.

2.5电镜样品准备
用于TEM分析的冰激淋样品（大约100 mm3）在-25℃下取样，用外科手术柳叶刀将样品放入-196℃液氮中，冰激淋变成＜1.0 mm3大小的颗粒。将冰冻的颗粒转移到含有3%(v/v)的戊二醛无水甲醇溶液中，在-80℃下放置4天。再将样品在-40℃下放置1天，然后转移到-20℃下放置2天。先用-20℃预冷的无水甲醇洗固定好的样品，然后用-20℃预冷的无水乙醇洗。然后依次用体积比3：1的甲醇/ LR-金色树脂（LR-gold resin），1：1的甲醇/LR-金色树脂和1：3的甲醇/ LR-金色树脂洗，最后用100%的LR-金色树脂洗。然后将样品放入含0.1%(w/v)二苯（基）乙二酮(benzyl)的LR-金色树脂的胶囊里过夜。LR-金色树脂在-20℃ 360nm UV灯下浸透到样品里面发生聚合。将树脂块用LKB超微切片机（Leica Reichert Ultracut S,Vienna,Austria）切成90nm的薄片并立即放入用镍涂布的小格栏(Marivac Ltd., Halifax, NS, Canada)里。
2.6染色和电镜实验
采用Hillbrick,McMahon和McManus(1999)报道的方法进行免疫标记。阻滞剂为2%(w/w)牛血清白蛋白的20mM磷酸盐缓冲液（PBS,pH 7.4）。含有超薄切片的格栏用数滴阻滞剂漂洗20min以防止非特异性的抗体结合。用磷酸缓冲液将一抗（primary antibody）稀释200倍，在潮湿箱内将格栏内的超薄切片漂洗1h。空白用不含一抗的PBS漂洗。格栏用PBS洗4次接着用数滴PBS再洗（四次，每次2min）。为了确定一抗的位置，格栏再用稀释20倍（PBS稀释）的二抗(secondary antibodies)在室温下的潮湿箱内漂洗2h。格栏用PBS洗4次接着用数滴PBS再洗(洗4次，每次2分钟)，最后用数滴蒸馏水洗（洗4次每次2min）。格栏然后用2%(w/w)乙酸双氧铀(uranyl acetate)溶液染色10min以强化对比度。TME用LEO-912AB显微镜（Munster,Germany）在100kV下操作。图片用Proscan CCE摄像机和SIS Esivision软件公司的SIV（图像观看软件）观看。每个冰激淋样品至少做成三个树脂样品，每个树脂样品至少检测三个薄片而且每个薄片至少检测20个不同的区域。

英文原文
2.5. Sample preparation for transmission electron
microscopy (TEM)
Ice cream specimens (approximately 100mm3) for TEM were taken from the inner bulk of the hardened samples at-25℃with a surgical blade and immediately placed into liquid nitrogen (-196℃), where they were broken into o1mm3 pieces. The freeze-substitution technique was based on that of Goff et al. (1999).Frozen specimens of ice cream were transferred into vials that contained 3% (v/v) glutaraldehyde in absolute methanol, which had been kept at -80℃ for 4 days. Although minimal fixation is important for immunogold labeling, to prevent loss of antigenicity, the use of glutaraldehyde was possible due to the enhanced
sensitivity that arises from the use of polyclonal antibodies compared to monoclonal antibodies. Samples were transferred t-40℃ for 1 day, during which the fixative mixture melted, and gradual freeze substitution of ice with methanol and some fixation with
glutaraldehyde took place. Samples were then transferred to-20℃ for 2 days where fixation proceeded at a faster rate. The fixative mixture was replaced by washing the specimens in precooled (-20℃) 100% methanol followed by washing with absolute ethanol. Infiltration of specimens with resin was carried out by graded washing with a mixture of methanol and LR-gold resin at volume ratios of 3:1, 1:1 and 1:3, followed by washing in 100% resin. Specimens were placed into gelatin capsules filled with resin containing 0.1% (w/v) benzyl and held overnight. Then the resin-infiltrated samples were polymerized under 360 nm UV light at -20℃. Resin blocks were sectioned at a thickness of 90nm using an LKB ultramicrotome (Leica Reichert Ultracut S, Vienna, Austria). Sections were immediately mounted on nickel-coated grids (Marivac Ltd., Halifax, NS, Canada).
2.6. Immuno-gold labeling and TEM
Immunolabeling was based on the methods described by Hillbrick, McMahon, and McManus (1999). The blocking agent was comprised of 2% (w/w) bovine serum albumin in 20mm phosphate buffer saline (PBS, pH 7.4). Grids containing the sections were floated for 20 min on drops of blocking agent to prevent nonspecific binding of the antibodies. The grids were floated in a humidity chamber on the primary antibody diluted 1:200 with PBS for 1 h. The control was floated on PBS instead of the primary antibody. Grids were rinsed four times with PBS followed by floating on drops of PBS (four times at 2 min each). To localize the positions of the primary antibodies the grids were then floated on the secondary antibodies diluted 1:20 with PBS for 2 h in a humidity chamber at room temperature. Grids were rinsed with PBS (four times) followed by floating on drops of PBS (four times at 2 min each), and were finally washed by floating on drops of distilled water (four times at 2 min each). The grids were then stained with 2% (w/w) uranyl acetate solution for 10 min to enhance the contrast. TEM was performed with a LEO-912AB microscope (Munster, Germany) operated at 100 kV. The images were examined using a Proscan CCE camera and SIV (soft imaging viewer) from SIS Esivision software. At least three blocks of each ice cream sample were sectioned, and at least three sections per block and 20 fields per section were viewed.

太有趣了，做电镜还能买各种牌子的冰淇淋，真爽阿！！！

我是想要做透射电镜啊，请各位高手帮忙，看广州什么地方可以做。

华南理工大学就有啊。