General comments: Immunohistochemistry demands keeping a gentle balance between exposure of antigenic sites and preservation of structure. Some treatments might not be enough to reveal antigenic determinants, recognized by antibodies. Other treatments 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 your primary antibodies, from overnight to just few hours.
- Perform incubations with primary 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. Agrisera secondary antibodies can be found here.
Overview of steps in procedure
- 1 % paraformaldehyde in PBS, 10 min at RT
- 2 % paraformaldehyde + 0.2 % Triton for 25 min at RT
- 4 % paraformaldehyde in PBS for 20 min at RT, permeabilized with 0.2 % Triton X-100 in PBS for 20 min methanol: acetone (1:1) 15 min RT, 10 min rehydration in PBS
- 5 % formaldehyde, 30 min at RT
- 3.5 % formaldehyde in PBS for 10 min
- Antigen retrieved 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:
- li50 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
- 10 % normal goat serum + 2 % BSA + 0.2 % Triton X-100 in PBS for 20 min
- 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
0.2 % Triton X-100 in PBS
When using IgY antibodies, washing steps should be more extensive.
Examples of primary antibody incubation:
- 1 h 37°C
- PBS + 1 mg/ml BSA 2 h RT
- ON 4°C
Agrisera Matching Secondary antibodies
High quality blocking reagents
Suggested protocol for immunolocalization in plant tissue: Pasternak et al. (2015). Protocol: an improved and universal procedure for whole-mount immunolocalization in plants. Plant Methods.
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