How do you get antigen retrieval?
John Peck
Updated on March 30, 2026
Method
- Deparaffinize and rehydrate the sections.
- Add the appropriate antigen retrieval buffer to the microwaveable vessel.
- Place the slides in the microwaveable vessel.
- When 20 min has elapsed, remove the vessel and run cold tap water into it for 10 min.
- Continue with the immunohistochemical staining protocol.
What is heat induced epitope retrieval?
Heat induced epitope retrieval (HIER) may be defined in the simplest terms as the use of heat coupled with specific buffered solutions to recover antigen reactivity in formalin fixed paraffin embedded tissue. The HIER procedure has had a huge impact on the way IHC is utilized in the diagnostic process.
How does antigen retrieval work?
Antigen retrieval enables an antibody to access the target protein within the tissue. Masked epitopes can be recovered using either enzymatic/proteolytic antigen retrieval, or heat-induced antigen retrieval methods. In the enzymatic method, proteases such as proteinase K, trypsin, and pepsin are used.
What are the different methods of retrieval of masked antigens?
When discussing antigen retrieval methods, techniques generally fall into two main categories, protease-induced epitope retrieval (PIER) and heat-induced epitope retrieval (HIER). Once optimized, the effects of antigen retrieval can be pronounced.
Is antigen retrieval necessary?
Is antigen retrieval necessary on frozen tissue sections? Antigen retrieval on frozen tissue is not recommended. The retrieval process can be too harsh and damage the tissue. However, it is often recommended to restore antigenicity in formalin-fixed tissues.
Is antigen retrieval necessary for frozen sections?
Do I need antigen retrieval for frozen sections?
For frozen sections, antigen retrieval is not required as the fixation time with aldehydes is very short (10–30 minutes) and doesn’t allow the formation of cross-links.
How long does citrate buffer last?
Store this solution at room temperature for 3 months or at 4 C for longer storage. Note: this buffer is commonly used and works perfectly with many antibodies.
What is microwave antigen retrieval?
Antigen retrieval is commonly described as a ”high temperature heating method to recover the antigenicity of tissue sections that had been masked by formalin fixation” (Shi et al., 2001), but in some cases, the hidden epitopes can be recovered only with the detergent treatment, without heating (Brown et al., 1996).
What is IHC FR?
IHC-Fr. Immunohistochemistry (frozen sections) Fixation: immersion. Sample is frozen prior to sectioning.
Can you reuse citrate buffer?
Add more citrate buffer after the first 10 minutes, then microwave another six minutes. 3.9 Next, cool down the slides for 10 minutes. Pour the Citrate Buffer back into the original container to reuse.
How important are pH and temperature for the retrieval of antigens?
We conclude that the pH and temperature of the solutions and the irradiation time are of great importance for the retrieval of antigens, but in a different way for the two antibodies studied.
What is the optimal antigen retrieval time?
Optimal antigen retrieval time may vary as per the antibody specification; however, we have achieved the best results between 10–20 min for several antibodies such as CD 45, CYP 3 A, PCNA etc. Tissues can be boiled in this way even more than 30 min without any damage or loss from the slide.
What is the best method for heat-mediated antigen retrieval?
Many labs use a vegetable steamer or rice cooker for heat-mediated antigen retrieval. The procedure is similar to microwaving in that it maintains the temperature of the buffer at 100°C, but without the vigorous boiling of the microwave method. This method may be adapted to a water bath set at 100°C in place of the steamer.
How do I prepare antigen retrieval buffer for immunohistochemistry?
Antigen retrieval buffer (Tris/EDTA pH 9.0, sodium citrate pH 6.0, or other) A control experiment is recommended to optimize retrieval time, where slides of the same tissue section are retrieved for 1, 2, 3, 4 and 5 min before being immunohistochemically stained. Add the appropriate antigen retrieval buffer to the pressure cooker.
