A New Immunoelectron Microscopy Approach for the Detection of Immunoglobulin and Complement Deposits in E poxy-Embedded Renal Biopsies

Abstract

The purpose of this study was to examine the diagnostic value of a new immunoelectron microscopy technique (IEM) for detection of immunoglobulin and complement deposits in epoxy-embedded renal biopsies. Twenty-four renal biopsies were embedded in epoxy resin following a tissue processing involving moderately increased amount of accelerator, DMP-30 (Tri(Dimethyl Amino Methyl) Phenol), in the infiltration steps. Following antigen retrieval by heating in citrate buffer, immunogold labeling was performed on ultrathin sections from these epoxy blocks with antibodies against immunoglobulins and complement. The sections were counterstained with urnayl acetate and lead citrate without any enhancing procedures. The preservation of the ultrastructure with this method was similar to that usually seen in epoxy embedded material. The immunogold labeling was intense and distinct. Immunofluorescence (IF) for light microscopy was carried out on frozen sections of parallel tissue samples. The correspondence between IF and IEM were good, but in some cases higher sensitivity for IgA with IEM than IF was observed in the sense that smaller amounts of antigen were detectable with IEM. The combination of moderately increased amount of accelerator and antigen retrieval is superior to previous methods with respect to ease of use, ultrastructural preservation, and intensity of the immunolabeling. Moreover, the renal tissue can be processed in an automatic ultraprocessor together with other specimens which are to be prepared for routine electron microscopy.

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“…Unlike some other studies, we did not require further antigen retrieval to successfully immunolabel our epoxy sections. Previous studies have found that antigen retrieval by heating in citrate buffer (Stirling and Graff 1995;Brorson and Strøm 1998;Groos et al 2001), Tris-HCl (Yamashita et al 2009), or alkaline solutions (Yano et al 2003) significantly increased immunlabeling probe density over that obtained with etching alone. Other aspects, such as an increased amount of accelerator in epoxy resin (Brorson 1998a), the use of propylene oxide in specimen processing (Brorson 1996), the sectioning process itself (Hann et al 2001), and osmication of tissue (Brorson 2004), have also been shown to affect antigen retrieval from epoxy sections.…”