Protocol for immunocytochemistry

Solutions needed:.

• Phosphate buffer saline (PBS), pH 7.4
  
  
• 4 % Paraformaldehyd in PBS, pH 7.4
  
  
• 0.1 % saponin in PBS
  
  
• 50 mM NH₄Cl
  
  
• 3 % BSA in saponin/PBS
  
  
• Mowiol or DAKO
  

Procedure:

1. Rinse 6 coverslips in 70 % ethanol and flame.
   
   
2. Let cells grow on coverslips to desired density. Note that some cell lines require poly-lysine coated cover slips.
   
   
3. Transfer coverslips to 6 well plate.
   
   
4. Wash coverslips twice with PBS.
   
   
5. Fix cells with 3 ml/well paraformaldehyde solution for 15-20 min at RT.
   
   
6. Wash 3 times with 50 mM NH₄Cl.
   
   
7. Incubate for 5 min with 50 mM NH₄Cl.
   
   
8. Incubate for 10 min with saponin/PBS.
   
   
9. Incubate for 30 min with BSA/saponin/PBS.
   
   
10. Put a piece of parafilm on wet Whatman paper and apply 200 µl of primary antibody solution in BSA/saponin/PBS (appropriate dilution must be determined experimentally).
    
    
11. Put coverslips upside down on antibody-solution and incubate for 1 h at RT.
    
    
12. Transfer slips to 6 well plate and wash twice with saponin/PBS.
    
    
13. Incubate for 10 min with BSA/saponin/PBS.
    
    
14. Repeat steps 10-13 with secondary antibody (optimal dilution must be determined experimentally). Avoid bright light when working with the secondary antibody to minimize photo bleeching of the fluorescent dye.
    
    
15. Wash twice with saponin/PBS.
    
    
16. Wash twice with PBS.
    
    
17. Add a drop of Mowiol or DAKO fluorescent mounting medium on slide and mount dry coverslip.
    
    
18. Incubate for 30 min at RT.
    
    
19. Incubate over night at 4°C.
    
    
20. Incubate for 30 min at RT.
    
    
21. Seal with nail polish and microscope.