Immunohistochemistry FAQ

FAQS 

Immunohistochemistry

1. Where 'including paraffin-embedded sections' is not included in a description, does it mean that the antibody has been tested and found not to work, or does it mean that it has never been tested?

No, it does not mean the antibody has been tested and found not to work. It is certainly worth trying it. For testing an antibody's suitability, we recommend performing antigen retrieval on the sections by heat treatment and using the antibody at a starting dilution of 1/50 (8ug IgG/ml). 

 

2. If an antibody can be used for paraffin-embedded sections, what method of antigen retrieval should I use?

As a starting point, we recommend using sodium citrate buffer with heat treatment (95°C). However, it is possible that alternative methods (e.g. enzymatic digestion) may be better for your sections. We recommend each researcher optimises the antigen retrieval conditions for their own tissues. 

 

3. What fixation method is best and what dilution of primary antibody should I use?

For immunofluorescence, we recommend methanol fixation of the cells and a starting dilution of 1/50 (8ug/ml). For frozen sections, we recommend acetone fixation of the sections and a starting dilution of 1/100 (4ug/ml). For formalin-fixed, paraffin-embedded sections, we recommend antigen retrieval using heat treatment and a starting dilution of 1/50 (8ug/ml).

To determine the optimal dilution required, we recommend that each researcher perform a serial titration against relevant positive and negative controls.