Antigen Retrieval Using Microwave Heating

Wild-Type p53 Antigen Retrieval Using Microwave Heating 

Tissues are fixed with 10% neutral phosphate-buffered formalin and embedded in paraffin, and sections (5 pm thick) are mounted on poly-L-lysine-coated slides heated at 60°C for 30 min. The sections are deparaffinized in four changes of xylene and then rehydrated in a descending series of ethanol. Endogenous peroxidase activity is quenched by immersing the sections in 1% H202 in distilled water for 5 min. The sections are rinsed in three changes of distilled water, transferred to a moist chamber, and covered with PBS. 

The slides are placed in 0.1 M sodium citrate buffer (adjusted to pH 6.0 with NaOH) in a plastic jar, which is transferred into a microwave oven. They are heated at 750 W for 17 min, with brief interruptions at 7 and 12 min to replace evaporated volume with distilled water. The slides are cooled to room temperature in the citrate buffer and transferred to PBS. Nonspecific background staining is blocked by treating the sections with diluted 

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GENERAL PROCEDURE FOR ANTIGEN RETRIEVAL USING MICROWAVE HEATING... 

Frozen sections, as well as sections of formaldehyde-fixed and paraffin-embedded tissues, can be used for direct or indirect immunofluorescence staining after antigen retrieval using microwave heating. Although frozen sections yield higher sensitivity than that obtained with paraffin sections, the latter approach is necessary for retrospective studies of archival specimens. Moreover, fresh tissue is not always available. 

Hayat, M. A. 1999. General procedure for antigen retrieval using microwave heating. Microsc. Today 99(7) 28-30. 

Immunohistochemical detection of wild-type and mutant p53 proteins can be carried out in fresh-frozen as well as formalin-fixed and paraffin-embedded tissues. This is best accomplished by using antigen retrieval with microwave heating or other types of heating such as autoclaving. A number of monoclonal antibodies are commercially available, the characteristics and sources of which are listed in Tables 10.3 and 10.4 and on pages 50-51. 

Hazelbag, H. M., V. D. Broek, L. J. C. M., Van Dorst, E. B. L., Offerhaus, G. J. A., Fleuren, G. J., and Hogendoom, P. C. W. 1995. Immunostaining of chain-specific keratins in formalin-fixed, paraffin-embedded tissues a comparison of various antigen retrieval systems using microwave heating and proteolytic pretreatments. J. Histochem. Cytochem. 43 429-437. 

In this book chapter, a standardized and efficient protocol for a reliable antigen retrieval of intracellular Ap in AD mouse models is described in detail using microwave heating and, especially, crucial formic acid (FA) pretreatments. The described antigen retrieval using FA is compatible with different signal detection methods and has been quantitatively evaluated in the APP/PSIKI mouse model. The outcome was corroborated in three other AD mouse models, in which intraneuronal Ap was found to accumulate [25],... 

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. 

In contrast to microwave heating, steam treatment heats slides slowly to a uniform temperature. This avoids boiling the antigen retrieval fluid and minimizes section detachment from slides. Steam heat used in combination with EDTA and protease digestion has been... 

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