Heat-induced Antigen Retrieval in Conventionally Processed Epon-embedded Specimens

Abstract

SummaryWe studied the effectiveness of heat-induced antigen retrieval (HIAR) in conventionally processed, epon-embedded specimens and the mechanisms of HIAR in the specimens. Frozen sections were first immunostained to examine the possibility of using HIAR for 18 antigens to avoid the effects of epoxy resin embedment. The antigenicity of 7 out of 18 antigens was retrieved with glutaraldehyde fixation followed by osmium tetroxide treatment whereas none were retrieved with glutaraldehyde fixation without post-osmication. Six antigens also exhibited positive immunostaining in semi-thin epon sections when the sections were deplasticized with sodium ethoxide followed by autoclaving. In the immunoelectron microscopy with the post-embedding method, positive reactions with fine ultrastructures were obtained using HIAR without deplasticization. These results suggested that osmium tetroxide binds to ethylene double bonds (which are introduced into protein crosslinks by glutaraldehyde) and forms an extremely stable resonance interaction with the Schiff bases, thus destabilizing the protein crosslinks. Heating also further degrades these crosslinks. The present study demonstrated that archival epon blocks can be useful resources for immunohistochemical studies for both light and electron microscopy. (J Histochem Cytochem 62:584-597, 2014) Keywords heat-induced antigen retrieval, osmicated and epon-embedded specimen, immunoelectron microscopy, mechanism of antigen retrieval

The Peculiar Pattern of Type IV Collagen Deposition in Epiretinal Membranes

“…Then, slides were washed in ethanol and distilled water and incubated with 20% donkey serum additioned with 0.3% Triton X-100. Heatinduced antigen retrieval (HIAR) was carried out according to Yamashita and Okada, 23 autoclaving slides in 0.1M Tris (pH 9.0) at 120° for 10 minutes. Before incubating slides with each primary antibody, sections were always treated with 5% bovine serum albumin (BSA) in phosphate buffered saline (PBS) while before applying each secondary antibody, sections were incubated with 10% non-immune serum belonging to the animal species employed to raise the antibody.…”

“…In order to retrieve antigenicity, ultrathin sections mounted on Neoprene W-coated nickel grids were placed for 60 minutes at 95° in 0.5M Tris (pH 9.0) according to Yamashita and Okada. 23 Unspecific binding sites were quenched with 0.2% sodium borohydride for 10 minutes and, after extensive rinsing, with 1% ovalbumin in PBS. Subsequently, grids were incubated overnight at 4°C with anti-Col IV antibody (dilution 1:50).…”

Correlative Light-Electron Microscopy (CLEM) and 3D Volume Imaging of Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Langerhans Islets

“…Precise mechanisms of (HIAR) is that the extended polypeptides by heating are charged negatively or positively at basic or acidic pH and that an electrostatic repulsion force acts to prevent random entangling of polypeptides caused by hydrophobic attractive force and to expose antigenic determinants, during cooling process of HIAR solution. HIAR is a powerful tool to all types of immuno-EM for antigen-antibody assay [54][55][56].…”

“…However, the weak tissue antigenicity of Epon-embedded sections poses a problem for immunoassays. There have been attempts to improve immunolabelling of epoxy sections by etching hydrophobic Epoxy resin with different alkali solutions [66], retrieving antigenicity by sodium metaperiodate [21,67], protease treatments [68], or heating (autoclaving or microwaving) thick or ultrathin sections for immunoelectron microscopy with various salt solutions [45,56,[69][70][71]. In the present study, in addition to etching the hydrophobic Epon, we utilised autoclaving and pretreatment with hydrogen peroxide to enhance endocrine peptide immunoreactivity.…”

Methyl methacrylate embedding to study the morphology and immunohistochemistry of adult guinea pig and mouse cochleae

“…To enhance the immunogenicity of samples embedded in paraffin or resin, retrieval techniques are widely used. High temperature antigen retrieval in different solutions (Shi et al, 2011;Yamashita and Okada, 2014) indeed leads to improved antigenicity, but it renders the protocol longer and more complicated, and it is disadvantageous for tissue preservation.…”