Antigen Retrieval Techniques

Abstract

S U M M A R Y Development of the antigen retrieval (AR) technique, a simple method of boiling archival paraffin-embedded tissue sections in water to enhance the signal of immunohistochemistry (IHC), was the fruit of pioneering efforts guided by the philosophy of rendering IHC applicable to routine formalin-fixed, paraffin-embedded tissues for wide application of IHC in research and clinical pathology. On the basis of thousands of articles and many reviews, a book has recently been published that summarizes basic principles for practice and further development of the AR technique. Major topics with respect to several critical issues, such as the definition, application, technical principles, and further studies of the AR technique, are highlighted in this article. In particular, a further application of the heat-induced retrieval approach for sufficient extraction of nucleic acids in addition to proteins, and standardization of routine IHC based on the AR technique in terms of a test battery approach, are also addressed. Furthermore, understanding the mechanism of the AR technique may shed light on facilitating the development of molecular morphology. Immunohistochemistry (IHC) has created a wide field for functional (analytical or molecular) morphology, particularly since it has rendered immunoperoxidase methods applicable to routine formalin-fixed, paraffin-embedded tissues based on a series of technical developments. These include increasingly sensitive detection systems and several pretreatments before the immunostaining procedure to recover antigenicity masked by formalin fixation. However, the growing interest of pathologists who attempt further to expand the application of IHC staining on formalin-fixed, paraffin-embedded tissue sections were frustrated by inconsistent results on fixed tissues. More than two decades ago, various alternative fixatives were tried in an attempt to replace formalin in an irreversible chemical reaction of formalin-protein, but they have failed, and it is likely that an ideal fixative will never be found (Larsson 1988). On the other hand, early IHC methods, including initial unmasking techniques such as enzymatic digestion, failed to yield satisfactory immunostaining for many antigens. Therefore, the search for a simple and effective retrieval method has become a hot topic in IHC since the early 1970s (Taylor and Cote 1994). In response to the need for a more effective method to recover the formalin-modified antigenicity, a high-temperature-heating antigen retrieval (AR) technique, the method of boiling paraffin tissue sections in water, was shown 10 years ago to rendering IHC staining possible on archival formalin-fixed, paraffin-embedded tissue sections (Shi et al. 1991). This AR technique was promptly accepted and employed by pathologists and morphologists worldwide, serving as a simple and effective method to achieve satisfactory immunostaining on archival tissue sections, enthusiastically described as "a revolutionary new technique" and "breakthrough in pathology" (Gown et ...

A nondestructive molecule extraction method allowing morphological and molecular analyses using a single tissue section

“…22 So far, the mechanisms of crosslinking reversal as the base for antigen retrieval are not well understood. [23][24][25] Elucidation of these mechanisms will be very helpful for optimization of macromolecular extraction. Current methods for antigen retrieval include proteinase predigestion, 26 chemical pretreatment, [27][28] and heat-induced epitope retrieval.…”

Vitamin D receptor distribution in intestines of domesticated sheep Ovis ammon f. aries

“…Sections were dewaxed in xylene and isopropyl alcohol, hydrated through serial dilutions of ethanol to water and rinsed in trizma-buffered saline for 2 min (buffer stock solution: 6.1 g trizma base, 50 ml H 2 O and 37 ml 1 N HCl, diluted with H 2 O to 1000 ml solution, adjust pH to 7.6; working solution: 100 ml buffer stock solution plus 900 ml saline, 0.85 %). Antigen retrieval (Shi et al, 2001) was carried out by incubating the sections in citrate buffer (0.01 mol l -1 , pH 6.0; ProTaqs ® citrate buffer concentrate and diluted with H 2 O to finally obtain 1000 ml solution; Medite, Nunningen, Switzerland) in a microwave oven (600 W, 3 X 5 min). Sections were cooled down to room temperature in citrate buffer for 20 min and endogenous peroxidases inhibited using 3% peroxide for 10 min followed by incubation in TBS for 5 min.…”

The role of humoral autoimmunity in gastrointestinal neuromuscular diseases

“…formalin cross-links) with antigen retrieval. A number of techniques such as pressure cooking, protease treatment, and microwaving in an appropriate buffer may be employed, although the molecular mechanisms underlying the process are poorly understood (Shi et al 2001).…”