Antigen retrieval methods

Yano S, Kashima K, Daa T, et al. An antigen retrieval method using an alkaline solution allows immunoelectron microscopic identification of secretory granules in conventional epoxy-embedded tissue sections. /. Histochem. Cytochem. 2003 51 199-204. 

Namimatsu S, Ghazizadeh M, Sugisaki Y. Reversing the effects of formalin fixation with citraconic anhydride and heat a universal antigen retrieval method. J. Histochem. Cytochem. 2005 53 3-11. 

Evers P, Uylings HB. An optimal antigen retrieval method suitable for different antibodies on human brain tissue stored for several years in formaldehyde fixative. I. Neurosci. Methods 1997 72 197-207. 

Jiao Y, Sun Z, Lee T, et al. A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections./. Neurosci. Methods 1999 93 149-162. 

Ramos-Vara J, Beissenherz M. Optimization of immunohistochemical methods using two different antigen retrieval methods on formalin-fixed, paraffin-embedded tissues experience with 63 markers. I. Vet. Diagn. Invest. 2000 12 307-311. 

Figure 20.3 SDS-PAGE of proteins extracted from archival fresh-frozen (lane 2) and FFPE (lane 3) GBM tissue using the antigen retrieval method. Reproduced with permission from Reference 14.

This is an important observation given that plasma membrane proteins are often used as markers of disease. This experiment demonstrated that shotgun proteomic analysis could be successfully performed on microdis-sected, formalin-fixed tissues using the antigen retrieval method with a sensitivity equal to that of analysis of the soluble fraction of a fresh-frozen sample. 

Hyatt MA (2002) Microscopy, Immunohistochemistry, and Antigen Retrieval Methods For Light and Electron Microscopy. Plenum, New York... 

Standard MWO Antigen Retrieval Method—Basic Method (5,6,15,17,19)... 

Brown, C. Antigen retrieval methods for immunohistochemistry. Toxicol. Pathol. 

Also, different forms of an antigen require different concentrations of the antibody for their maximal detection. This is exemplified by the PC-10 primary antibody, which identifies PCNA antigen at a dilution of 1 1000 in epithelial cells in normal colon tissue, whereas a dilution of 1 400 is required to localize these proliferating cells in adenomatous polyps (Holt et al., 1997). In contrast, some types of antigens (e.g., Ki-67) can be optimally detected in various tissue types at the MIB-1 dilution of 1 50, using the microwave heating antigen retrieval method. However, in a few studies MIB-1 dilutions of 1 20 to 1 100 have been used. 

There are many reasons for the lack of consensus on this highly complex phenomenon, and they are discussed below. Various studies mentioned above were conducted using different parameters of antigen retrieval methods, including antigen retrieval fluids, pH, heat source, temperature, and duration of treatments for detecting different antigens. The type of fixation and duration of fixation also varied in these studies. Other variants were the type of epitope and antibody and source of antibodies used. 

As stated earlier, lack of a standardized antigen retrieval method results in intra- and interlaboratory variability in immunostaining results. The following method of microwave... 

The following recent comparative studies demonstrate that no single antigen retrieval method is optimal for all types of antigens. 

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