What is antigen retrieval technique
Tissue sections pretreated with the antigen retrieval reagents that break the protein cross-links formed by formalin fixation and thereby uncover hidden antigenic sites.
Which antigen retrieval method should I use for a specific antibody
Some companies test their antibodies for immunohistochemistry application and will include a suggested antigen retrieval method in the datasheet (note: some of the suggested method is not optimal). If no one of above gives you a clear answer, a test is needed.
How to set up a test for a new antibody with various antigen retrieval methods
Initially, you may want to test two methods from HIER such as citrate buffer (pH 6) and Tris-EDTA (pH 9), and one or two methods from PIER such as Proteinase K and/or Trypsin. This should give you a good chance to obtain an optimal one for a specific antibody.
I have tested my antibody with various antigen retrieval methods, but still no results
The antibody may not be appropriate for formalin-fixed, paraffin-embedded tissues (some antibodies only work for frozen sections) or the antibody has a problem (expired or bad) and you may need to switch to a different antibody.
In heat induced epitope retrieval (HIER) methods, which one is the best
Citrate buffer (pH 6) method is one of the commonly used. Some studies showed that Tris-EDTA (pH 9) or Tris-HCl (pH 10) is superior. A test is probably the best way to know which one is the best for a specific antibody.
What are key factors to be considered when I do pretreatment with HIER
(1) Temperature of retrieval solution should be around 95 °C.
(2) Incubation time should be at least 10 minutes and it is usually around 20 minutes.
(3) pH value of retrieval solution is depending on which solution you are using.
In proteolytic enzyme induced epitope retrieval (PIER) methods, which one is the best
Proteinase K seems to be the commonly used one. The concentration, incubation time and temperature probably are more important factors to consider. Again, a test is the best way to know which one is the best for a specific antibody.
What are key factors to be considered when I do pretreatment with PIER
(1) Concentration of enzyme is usually 0.05-0.1% depending on type of tissue and fixation.
(2) Incubation time could be 5-30 minutes and 10-15 minutes is commonly used.
(3) Incubation temperature is usually at 37 °C.
Do I need to do antigen retrieval for frozen sections: Antigen retrieval is not needed for immunostaining of fresh frozen sections. Because antigen retrieval is to break the cross-links formed by formalin fixation and fresh frozen sections are sectioned and fixed with ice cold acetone for 5-10 minutes.