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.
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.
我的社区
签到
赛默飞实验室产品焕新计划有奖调研！
【七月征文】不一YOUNG实验“猿”
报名开启：ICS2024第十三届光谱网络会议！
推荐收藏！盘点中药材及饮片检测解决方案
