Quantitative Evaluation of Preparation Procedures Affecting Immunogold Staining in Post Embedding Immunocytochemistry.

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Abstract

Effect of fixative concentration, fixation time, embedding media, condition by dehydration and embedding, and incubation time with primary antibody upon the labeling intensity, was investigated by quantitative immunoelectron microscopy. Rat liver tissue and erythrocytes processed under various conditions of preparation were embedded in LR White or Lowicryl K4M. As concentration of glutaraldehyde (GA) in fixative increased, labeling density decreased. In the tissue and cells fixed with fixative containing 4% paraformaldehyde and 0.25% GA for 5-10min, cell ultrastructure and antigenicity were well preserved. As fixation was prolonged, labeling density was lowered. In the materials embedded in acrylic resins, such as LR White and Lowicryl K4M, high labeling density was obtained, compared with that obtained from embedding in epoxy resins such as Epon 812, Durcupan and Quetol 651. Temperature difference between dehydration and embedding affected strongly the preservation of antigenicity. There was no difference in labeling densities between embedding at 23°C and -20°C, when tissue slices were dehydrated at 4°C. To saturate the reaction of primary antibody with antigen on thin sections incubation should be carried out over 8hr. From these results, we recommend the following points in immunoelectron microscopy; 1) perfusion fixation with 4% paraformaldehyde+0.25% GA for 5min to 10min, 2) minimized temperature difference between dehydration and embedding of tissue, 3) use of acrylic resins for embedding, and 4) over 8 hr-incubation with primary antibody.

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