Increased yield of immunogold labeling of epoxy sections by adding para-phenylendiamine in the tissue processing.
The purpose of this study was to examine if the presence of para-phenylendiamine (PPD) in the tissue processing could increase the yield of immunogold labeling of the epoxy sections. Renal swine tissue with glomerular immune complex deposits with reactivity against IgG was embedded in epoxy resin. PPD was added (1) at the beginning of the dehydration, (2) in the first step with propylene oxide, (3) in the beginning of the dehydration and in all steps with propylene oxide included the infiltration step where propylene oxide and epoxy resin are mixed, or (4) PPD was totally avoided. The tissue was embedded with two different combinations of accelerator. Immunogold labeling with anti-IgG was performed on both non-heated and heated ultrathin sections. The immunogold labeling on the heated sections which were based on processing with PPD in all steps (3) was about 55-65% higher than the corresponding labeling for epoxy sections processed in total absence of PPD (4). The immunolabeling was not significantly increased when the tissue was processed with PPD only in the start of the dehydration (1) or in the first step with propylene oxide (2). We believe that tissue processing with sufficient PPD contributes to reduce the co-polymerization between the antigens and the epoxy polymer in the same way as excess of accelerator does (Brorson and Skjørten, 1996a). The practical significance of this study provides better opportunities for increasing the immunogold labeling of epoxy sections by adding PPD in the tissue processing, and our result may inspire other researchers to develop even more efficient methods for controlling the copolymerization between antigens and epoxy resin.
Brorson
S H
SH
Department of Pathology, Ullevål Hospital, Oslo, Norway. s.h.brorson@ioks.uio.no
Halvorsen
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Lønning
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Slaattun
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Sletten
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Rashid
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D000818:Animals; D004852:Epoxy Compounds; D006046:Gold; D007672:Kidney Cortex / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron / Q000379:methods*; D010655:Phenylenediamines; D016613:Plastic Embedding / Q000379:methods*; D013552:Swine
D004852:Epoxy Compounds; D010655:Phenylenediamines; D006046:Gold; C029728:4-phenylenediamine