Immunohistochemistry using IgY

General comments: Immunohistochemistry demands to keep a gentle balance between exposure of antigenic sites and preservig of the structure. Some treatments might not be enough to reveal antigenic deteminants recognized by antibodies. Other can simply wipe them off. Therefore some adjustment of fixing technique is necessary for good results.

In case of increased background problems - general comments:

• Shorten incubations with IgY primary antibodies from over night to just few hours
  
• Perform incubations with primary IgY antibodies at room temperature instead of cold room
  
• Increase Tween concentration
  
• Modify blocking procedure
  
• Try pre-adsorption of antibodies on the material lacking protein of interest
  
• Use some blocking protein in primary and secondary antibody buffers
  
• Try secondary antibodies from another supplier
  

Overview of steps in procedure

Tissue fixing:

• 1 % paraformaldehyde in PBS, 10min, RT
  
• 2 %parafomaldehyde + 0,2 % Triton for 25 min at RT
  
• 4 % paraformaldehyde in PBS for 20 min at RT,permabilized with 0,2 % Triton X-100 in PBS for 20 min methanol: acetone (1:1) 15 min RT, 10 min rehyfratation in PBS
  
• formaldehyde 5 %, 30 min RT
  
• formaldehyde 3,5 % in PBS for 10 min
  
• antigen retrived using microwave treatment in 10 mM citrate buffer pH 6 followed by heat treatment (boiling 5 min) in TBS containing 10 mM DTT, followed by 2x10 min washes in TBS
  

Examples of antibody dilution buffer for both primary and secondary antibodies:

• 50 mM Tris/Cl pH 7,4, 154 mM NaCl, 0,1 % Tween 20 + 10 % FCS
  
• PBS + 3 % BSA + 0,1 % Tween 20
  
• PBS + 2 % goat serum
  
• Blocking buffer:
  

Blocking:

• 10 % normal goat serum + 2 % BSA + 0,2 % Triton X-100 in PBS for 20min
  
• PBS + 1 mg/ml BSA and 10 mM NaN3
  
• PBS + 10 % FCS 30 min to 1 h
  
• PBS + 10 % BSA
  
• PBS + 2 % BSA + 0,2 % Tween 20 + 10 % glycerol + 0,05 % NaN3
  
• PBS + 5 % BSA + 0,1 % Tween 20
  
• PBS+0,3 % Triton X-100, 0,3 % BSA + 5 % normal goat serum
  
• PBS + 5 % milk powder
  

Washing: generally extensive
0,2 % Triton X-100 in PBS PBS

Primary antibody incubation:

• 1 h 37�C
  
• PBS+ 1mg/ml BSA 2 h RT, ON 4�C
  

Secondary antibodies:

• Anti-IgY from donkey IgG (Fab) 1: 1000, Jackson ImmunoResearch 1 h37�C
  
• Anti-IgY from rabbit, (Promega)
  
• Anti-IgY from donkey (Interchim)
  
• Anti-IgY, goat (Aves Labs)
  
• Anti-IgY, rabbit, to heavy and light IgG chains (Pierce).
  
Note: to amplify the signal another secondary antibody was used: swine anti-rabbit IgG (DAKO)

In case of questions, you are welcome to contact AgriSera!