Protocol for immunohistochemistry - cryo sections

Solutions needed:.

• Fixation solution: 4 % formaline / 0.9 % NaCl / 0.5 % ZnCl₂
  
  
• 0.1 M Tris-HCl, pH 7.2
  
  
• 20 % sucrose in 0.1 M Tris, pH 7.2
  
  
• 0.1 M glycine buffered with Tris-base at pH 7.4
  
  
• 0.05 M Phosphate buffer, pH 7.2
  
  
• Blocking solution: 10 % goat serum / 0.3 % Triton-X 100 / 0.05 M phosphate buffer, pH 7.2 / 0.45 M NaCl
  

Procedure:

1. Fix tissue in fixation solution.
   
   
2. Rinse tissue in 0.1 M Tris-HCl, pH 7.2 and change buffer 3 times.
   
   
3. Incubate tissue for 12-18 h in 20 % sucrose, 0.1 M Tris, pH 7.2.
   
   
4. Freeze tissue with TISSUE-TEK using a cryostat.
   
   
5. Cut tissue (6-8 µ) and mount sections on poly-lysine coated slides.
   
   
6. Freeze the mounted sections at -20°C until use.
   
   
7. Allow frozen sections to dry at room temperature for 15 min.
   
   
8. Incubate for 30 min in 0.1 M glycine buffer.
   
   
9. Block for 1-2 h in blocking solution.
   
   
10. Incubate with primary antibody diluted in blocking solution for several hours at RT.
    
    
11. Wash with blocking solution for 30 min and change buffer several times
    
    
12. Incubate with secondary antibody diluted in blocking solution for 2-3 h at RT. Avoid bright light when working with the secondary antibody to minimize photo bleeching of the fluorescent dye.
    
    
13. Wash with blocking solution for 30 min and change buffer several times.
    
    
14. Wash with 0.05 M phosphate buffer for 30 min and change buffer several times.
    
    
15. Mount with DAKO fluorescent mounting medium.
    
    
16. Incubate for 30 min at RT.
    
    
17. Incubate over night at 4°C.
    
    
18. Incubate for 30 min at RT.
    
    
19. Seal with nail polish and microscope.