Exosome Block Preparation: A Protocol to Preserve Exosomes From Cultured Human Colon Cancer Cells
Transcrição
To fix the purified exosome pellet, add 1 milliliter of 2.5% glutaraldehyde in a 0.1 M sodium cacodylate solution at pH 7.0. Incubate the lipid bilayer vesicles for 1 hour at 4 degrees Celsius. Next, remove the fixative and rinse the pellets with 1 milliliter of a 0.1 M sodium cacodylate buffer solution at room temperature for 10 minutes.
Then, post-fix the samples with 1 milliliter of 2% osmium tetroxide for 1 hour at 4 degrees Celsius. Remove the fixative and rinse the exosome pellet three times with the 0.1 M sodium cacodylate buffer, changing the buffer every 10 minutes. Then, dehydrate the sample by shaking it for 10 minutes each in a series of graded acetone concentrations.
Next, mix up a solution of three parts acetone and one part of low viscosity embedding mixture. Replace the acetone with this mixture and incubate the sample for 30 minutes. Continue to increase the ratio of the low viscosity embedding medium, incubating for 30 minutes with each step.
Finally, incubate the exosome pellet in 100% of the low viscosity embedding mixture overnight at room temperature. The following day, embed the sample in pure low viscosity embedding mixture using an embedding mold and bake it for 24 hours at 65 degrees Celsius.
