1. Starting from OCT-cryopreserved blocks, cut 4μm slices on cryostat.
2. Fixate slices using acetone at 4 °C for 10 minutes.
3. Allow to dry at room temperature. Now you can start technique or store samples frozen.
4. Thaw slides or directly start technique after fixation. If frozen, leave them at room temperature for at least 30 minutes.
5. Wash with PBS buffer (pH 7.4) for 10 minutes.
6. Incubation with primary antibody at appropriate dilution for 1 hour at room temperature.
7. Wash with PBS buffer (pH 7.4) for 10 minutes three times.
8. Incubation with secondary antibody at the appropriate dilution for 1 hour at room temperature in the dark. Hereinafter, technique must be performed in the dark.
9. Wash with PBS buffer (pH 7.4) for 5 minutes three times.
10. Incubate with DAPI. [100 ng / ml]. for 15 minutes at room temperature.
11. Wash with PBS buffer (pH 7.4) for 10 minutes.
12. Mount a coverslip in an aqueous medium.
13. Samples are now ready to be observed under fluorescence microscopy either classic or confocal.
14. Slides must be stored at 4 ° C in the dark.
Submitted by: María Fraga, Spain