How to Examine the Antigen-damaging Effect of Sodium Ethoxide on Deplasticized Epoxy Sections

Abstract

The purpose of this investigation was to develop a method that could be used to estimate how damaging sodium ethoxide is to different antigens with respect to immunolabeling when epoxy sections are deplasticized. If we obtain weak labeling for an antigen on deplasticized epoxy sections, this might be caused by the damaging effect of the ethoxide solution. It is therefore interesting to develop a method to check if this really is the reason. Fibrin clots and tissues of human kidney and thyroid were embedded in LR White resin. Some thin sections from these specimen blocks were exposed to sodium ethoxide in the same way as epoxy sections are when being deplasticized. Other sections from the same blocks were not exposed to sodium ethoxide. Both categories of sections were immunogold-labeled with anti-fibrinogen, anti-thyroglobulin, anti-IgA, anti-IgG, or anti-IgM. The intensity of immunolabeling of sections treated with ethoxide was compared with the immunolabeling of corresponding sections that were not treated with ethoxide. No significant differences were found in immunolabeling for fibrinogen, IgA, IgG, and IgM. For thyroglobulin, the intensity was approximately 30% less in tissues that were exposed to sodium ethoxide. The practical significance of this method is that we easily can examine the degree to which a given antigen is affected by sodium ethoxide, which is the agent used for deplasticizing epoxy sections.

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

“…In tissues or proteins fixed with glutaraldehyde and osmium tetroxide, sodium metaperiodate treatment probably oxidizes the osmium additives completely and forms the 1, 2-dioles and cleaves the diols to aldehydes, thus inducing further degradation of the protein-glutaraldehyde co-polymers. The results of the present study support the hypothesis of Kawahara et al (1992;1997) concerning the mechanisms of fixation by glutaraldehyde.…”

“…Six antigens localized in frozen sections (other than PCNA) were also detectable in semi-thin epon sections; thus, sodium ethoxide treatment appears to conserve the antigenicity of most antigens. Similarly, Brorson (1997) demonstrated that the immunoreactivity of most antigens is not affected by sodium ethoxide treatment, as ultrathin sections prepared from specimens fixed with formaldehyde and embedded in LR-White, a sodium ethoxide resistant resin, showed no significant difference in immunostaining after sodium ethoxide treatment. Mar and Wight (1988) reported similar results using epon-embedded tissues fixed with a formaldehyde and glutaraldehyde mixture.…”

Quantum Dot Immunocytochemical Localization of Somatostatin in Somatostatinoma by Widefield Epifluorescence, Super-resolution Light, and Immunoelectron Microscopy

“…Etching or deplasticizing of sections to reveal antigenic epitopes is a critical step when attempting to immunolabel epoxy sections. Solutions of sodium ethoxide (Brorson 1997), sodium metaperiodate (Stirling and Graff 1995;Groos et al 2001), and 2-methoxyethyl acetate (MEA) (Brorson and Reinholt 2008) are commonly used. Sodium metaperiodate is a strong oxidizing agent and, in the present study, etching of the section surface for 40 min at room temperature was found to give an acceptable balance of labeling probe density with retention of adequate ultrastructural detail.…”