Difference between revisions of "NJU-China-notebook.html"
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For transmission electron microscopy analysis, the exosome samples were prepared as described above. Briefly, the exosome pellet was placed in a droplet of 2.5% glutaraldehyde in PBS buffer and fixed overnight at 4 °C. The exosome samples were rinsed 3 times in PBS for 10 min each and then fixed in 1% osmium tetroxide for 60 min at room temperature. Then, the samples were embedded in 10% gelatine, fixed in glutaraldehyde at 4 °C and cut into small blocks (less than 1 mm<sup>3</sup>). The samples were dehydrated in increasing concentrations of alcohol. Then, the samples were placed in propylene oxide and infiltrated with increasing concentrations of Quetol-812 epoxy resin mixed with propylene oxide for 3 h per step. Finally, the samples were embedded in pure, fresh Quetol-812 epoxy resin and polymerised at 35 °C for 12 h, 45 °C for 12 h, and 60 °C for 24 h. Ultrathin sections were cut using a Leica UC6 ultra-microtome and stained with uranyl acetate for 10 min and lead citrate for 5 min at room temperature. The samples were then observed with a transmission electron microscope (JEM-1010) at a voltage of 80 kV. </br></br> | For transmission electron microscopy analysis, the exosome samples were prepared as described above. Briefly, the exosome pellet was placed in a droplet of 2.5% glutaraldehyde in PBS buffer and fixed overnight at 4 °C. The exosome samples were rinsed 3 times in PBS for 10 min each and then fixed in 1% osmium tetroxide for 60 min at room temperature. Then, the samples were embedded in 10% gelatine, fixed in glutaraldehyde at 4 °C and cut into small blocks (less than 1 mm<sup>3</sup>). The samples were dehydrated in increasing concentrations of alcohol. Then, the samples were placed in propylene oxide and infiltrated with increasing concentrations of Quetol-812 epoxy resin mixed with propylene oxide for 3 h per step. Finally, the samples were embedded in pure, fresh Quetol-812 epoxy resin and polymerised at 35 °C for 12 h, 45 °C for 12 h, and 60 °C for 24 h. Ultrathin sections were cut using a Leica UC6 ultra-microtome and stained with uranyl acetate for 10 min and lead citrate for 5 min at room temperature. The samples were then observed with a transmission electron microscope (JEM-1010) at a voltage of 80 kV. </br></br> | ||
For confocal microscopy analysis, exosomes (100 μg) loaded with fluorescence-labelled siRNA were incubated with the Neuro2A cells (10<sup>6</sup> cells). After 6 hours, the cells were washed, fixed and observed under a confocal microscope (FV 1000; Olympus, Tokyo). Pictures were taken under the following conditions: objective lens: PLAPON 60 × O; NA: 1.42; scan mode: XY; excitation wavelength: 405 nm for Hoechst 33342 and 555 nm for Alexa Fluor 555; and image size: 1024 × 1024 pixels. | For confocal microscopy analysis, exosomes (100 μg) loaded with fluorescence-labelled siRNA were incubated with the Neuro2A cells (10<sup>6</sup> cells). After 6 hours, the cells were washed, fixed and observed under a confocal microscope (FV 1000; Olympus, Tokyo). Pictures were taken under the following conditions: objective lens: PLAPON 60 × O; NA: 1.42; scan mode: XY; excitation wavelength: 405 nm for Hoechst 33342 and 555 nm for Alexa Fluor 555; and image size: 1024 × 1024 pixels. | ||
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Exosomes (100 μg) loaded with MOR siRNAs were incubated with Neuro2A cells (10<sup>6</sup> cells). After 24 h incubation, the recipient cells were collected for total RNA extraction and subsequent quantitative RT-PCR analysis of MOR siRNA and MOR mRNA, and for total protein isolation and subsequent western blotting analysis of MOR protein. | Exosomes (100 μg) loaded with MOR siRNAs were incubated with Neuro2A cells (10<sup>6</sup> cells). After 24 h incubation, the recipient cells were collected for total RNA extraction and subsequent quantitative RT-PCR analysis of MOR siRNA and MOR mRNA, and for total protein isolation and subsequent western blotting analysis of MOR protein. |
Revision as of 11:28, 18 September 2015
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Notebook
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