Encyclopedia of Experiments: Immunology
È necessario avere un abbonamento a JoVE per visualizzare questo Contenuto. Accedi o inizia la tua prova gratuita.
Transcript
To begin, fill some of the wells of a fresh 96-well plate with 100 microliters of washing solution. Transfer the sections to be analyzed from the storage plate to the one containing the washing solution. Leave the sections in the washing solution for 10 minutes at room temperature. Then, transfer each section into an empty well containing the same washing solution, and repeat the wash. Next, add a washing solution to the sections, and transfer them into new wells containing 100 microliters of blocking solution. Incubate at room temperature for at least 90 minutes and a maximum of 4 hours.
Dilute the primary antibodies, anti-PGP9.5 and anti-5G4 in the working solution. Transfer the sections into new wells containing 100 microliters of the working solution of the primary antibodies and incubate overnight at room temperature.
The next day, wash the sections in wells containing washing solution as previously described. Dilute the secondary antibodies - goat anti-rabbit to detect anti-PGP9.5 and goat anti-mouse to detect 5G4 in the working solution.
Transfer the washed sections into new wells containing 100 microliters of the working solution of the secondary antibodies. Cover the plate with aluminum foil to avoid the bleaching of fluorophore conjugated with secondary antibodies, and incubate at room temperature for 90 minutes.
After this, wash the sections two times in washing solution as previously described. Transfer the washed sections into new wells containing 100 microliters of DAPI for 5 minutes at room temperature. Wash the sections two times in washing solution as previously described. Then, mount the sections on a slide in the correct position, avoiding misfolding.
Add a few drops of mounting medium to the slide and cover the section with a coverslip. Let the slides dry overnight. The next day, store the slides in an appropriate box at 4 degrees Celsius, making sure to avoid light exposure.
Using an inverted fluorescence microscope or a confocal microscope, view the sections. Acquire the images by a camera connected to the microscope. Then, use a fluorescence microscope or a confocal microscope to analyze positive signals in sections, in terms of spatial distribution and intensity of the signal, as outlined in the text protocol.
