Mechanisms of Heat-induced Antigen Retrieval: Analyses In Vitro Employing SDS-PAGE and Immunohistochemistry

Abstract

S U M M A R YIn this study, we examined the mechanism of heat-induced antigen retrieval using analytical procedures involving SDS-PAGE, Western blotting, and immunohistochemistry. Five proteins were treated with 4% formaldehyde in the presence or absence of 25 mM CaCl 2 , then heated under various conditions after removal of formaldehyde and analyzed on SDS-PAGE. Formaldehyde produced inter-and intramolecular cross-links in the proteins. Heating at high temperatures cleaved these cross-links at all pH ranges examined (pH 3.0, 6.0, 7.5, 9.0) and produced almost the same electrophoregrams as the native proteins. Proteins treated with formaldehyde containing CaCl 2 showed similar electrophoretic patterns, observed without heating or after heating at pH 6.0 and pH 9.0 in the presence or absence of 10 mM EDTA. Western blot analyses demonstrated that the soluble forms of ␤ -actin (monomer and oligomers) and fibronectin were present in extracts from deparaffinized mouse uterine sections autoclaved for 15 min but not in extracts from unheated specimens. Nine of ten antigens, independent of their isoelectric points, exhibited much stronger immunoreaction in the sections heated at pH 9.0 than in those heated at pH 6.0. The second heating at pH 6.0 significantly decreased the immunostaining of the antigens that had been boiled at pH 9.0, but the immunostaining was recovered after a third heating at pH 9.0. These results suggest that the main mechanism of heat-induced antigen retrieval is disruption of the cross-links and that pH is an essential factor for a proper refolding of epitopes.

PH or Ionic Strength of Antigen Retrieval Solution: A Potential Role for Refolding during Heat Treatment

“…This HIAR pattern may correspond to the type -B pattern (V -form type) of the classifi cation by Shi et al 15 Four antibodies produced the second immunostaining pattern, which consisted of strong immunostaining when heated in TB (pH 9.0 and pH 10.5) (Fig. 17.1 ), corresponding to the type -C pattern (ascending type) described by Shi et al 15 The α -amylase immunostaining pattern was almost constant regardless of the pH value of the buffers used for HIAR when the antiserum was diluted 5,000 -fold, 18 but the type -B pattern was observed when the antiserum was diluted 50,000 -fold. 17 If highly diluted antibodies were to be used in the immunohistochemical studies described by Shi et al, 15 the type -A pattern (stable type) might become nearly equivalent to the type -B pattern.…”

Comparative analysis of two immunohistochemical methods for antigen retrieval in the optical lobe of the honeybee Apis mellifera: Myosin-v assay

“…These factors and others, such as molarity and the chemical composition of the AR solution, can act to standardize this method, as was shown in other studies 13,35,37,38 .…”

“…Otherwise, the presence of sodium chloride in the AR solution can preserve the structure of the polypeptides 35 .…”

Study of Formalin Fixation and Heat‐Induced Antigen Retrieval

“…10,28,31 Further, while formalintreatment provides thermal stability to RNase A, this stabilization is not suffi cient to prevent thermally induced protein denaturation at temperatures ( ≥ 100 ° C) typically used in heat -induced AR methods. 32 -34 The implications of this fi nding for the mechanism of AR will be discussed further in Section 15.6 .…”