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|
| 1. |
Prepare sterilized coverslip. |
| 2. |
Put them in the tissue-culture plate. |
| 3. |
Place and maintain the cell suspension in the tissue-culture dish for 24 hours until the cells are seeded onto the glass. |
| 4. |
Prepare fixative with fresh 4% paraformaldehyde solution in PBS. |
| 5. |
Wash coverslip twice with PBS. |
| 6. |
Remove PBS but keep the specimen from drying completely. |
| 7. |
Fix the specimen at room temperature by dipping in 4% paraformaldehyde solution for 10 minutes. |
| 8. |
Wash twice with PBS. |
| 9. |
Incubate them at room temperature in 0.1% triton x-100 or NP -40 in PBS to make them prone to infiltration. Incubation time will differ up to 15 minutes depending
on the types of antigen. |
| 10. |
Wash 5 minutes or longer with PBS 4 times. |
| 11. |
Place the coverslip on the even surface and keep submerged for 30 minutes in 5% BSA in PBS solution for blocking. |
| 12. |
Treat sufficient amount of diluted primary antibody at room temperature for an hour. |
| 13. |
Wash 5 minutes or longer with PBS 3 times. |
| 14. |
Treat Fluorochrome-conjugated anti-immunoglobulin antibody (labeled secondary reagent) at room temperature for 30 minutes (make sure it doesn't exceed 60 minutes). |
| 15. |
Wash 5 minutes or longer with PBS 3 times. |
| 16. |
After mounting using fluorescent mounting solution, keep it in the dark. |
