Immunocytochemistry

Immunofluorescence staining for neural cysts

1. Wash cells with chilled, filtered PBS twice.
2. Fix cystic tissues with 4% PFA for 1 hours at room temperature.
3. Wash tissues in PBS 3 X 5 minutes.
4. Add 0.1% SDS (dissolved in PBS) solutionto permeabilize cell membrane at room temperature for 3 hours.
5. Wash tissues with PBS for three times, each time 5 minutes.
6. Add 10% goat serum solution or 4% donkey serum solution (diluted in PBS) to block nonspecific binding at 4°C overnight
7. Dilute the primary antibody with blocking solution according to its datasheet. Add the primary antibody into the blocking solution; incubate for 24 hr at 4°C.
8. Wash tissues with PBS for 3 X 5 minutes at room temperature.
9. Add DAPI and the fluorescent-labeled goat- or donkey-raised secondary antibodies in blocking solution; incubate for 24 hr at 4°C. (Note: samples need to be protected from light during the following steps).
10. Wash tissues with PBS for 3 X 5 minutes at room temperature.
11. Pipette appropriate amount of 90% glycerol solution(in PBS) drop on a microscope slide gently, avoiding air bubble present.
12. Place the cover slip with samples gently onto glycerol solution drop, avoiding air bubble.
13. Wipe the glycerol solution overflow from edge.
14. Mount cover slip on microscope slide with transparent nail polish that can be quickly dried, dry overnight.
15. Samples are ready for imaging under microscope.

All primary antibodies, their sources, and dilutions are listed in table below.

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