A simple post-embedding system for the rapid demonstration of tissue antigens under the electron microscope

Abstract

A simple and versatile technique for the preparation of ultra-thin sections, which can be stained immunohistochemically directly on electron microscope grids, is presented. An anti-hapten immunoperoxidase procedure has been adapted for use on tissue fixed in a purified monomeric glutaraldehyde--picric acid mixture, and embedded in 'L R White', a recently formulated plastic resin. This plastic tolerates the use of partial dehydration of tissue, resulting in higher antigenic yields. In addition, no etching of ultra-thin sections is necessary, and the whole immunostaining procedure can be completed in less than 2 h. A comparison of commonly used fixatives is discussed. High-resolution micrographs showing general staining (uranyl acetate--lead citrate) of rat pancreas, and immunostaining of insulin and TSH in storage granules in perfusion-fixed rat tissue and of lambda-chain immunoreactive cells in immersion-fixed human tonsil are included as examples.

Highly sensitive immunodetection of DNA on sections with exogenous terminal deoxynucleotidyl transferase and non-isotopic nucleotide analogues.

“…Some 1.6% glutaraldehyde-fixed cells were acetylated as previously described (Wassefet al, 1979). After washing in Sorensen's buffer, cells were dehydrated through graded ethanol solutions and then processed for embedding in either epon or Lowicryl K4M using the technique of Roth et al (1981), or in LR White using the technique of Newman and Jasani (1984). Ultra-thin sections of the Various blocks were collected in platinum rings (diameter 4 mm) formed by a platinum wire (0.1 mm diameter; SA Johnson Matthey NV, Brussels, Belgium) and stored on distilled water until use.…”

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

“…One important feature has to be noted that no osmotic eVects can take place (Studer et al 1992). Epoxy resins (Matsko and Müller 2005) are preferentially used for morphological analysis, and methacrylates for immunolabeling (Acetarin et al 1986;Carlemalm et al 1982;Newman and Hobot 1987;Newman et al 1983;Roth et al 1981a;Scala et al 1992). Important to know: the freezesubstitution protocol (and there may exist about 100 diVerent ones) determines contrast formation.…”

Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level

“…Thus, the chemical reactivity of the cured resin, its method of curing and the degree of crosslinking are important parameters (Causton, 1984). Acrylate resins, such as Lowicryl K4M (Roth et al, 1981;Armbruster et al, 1982Armbruster et al, , 1983Carlemalm et al , 1982Carlemalm et al , , 1985Altman et al, 1984;Kellenberger et al, 1987) and LR White (Newman et al, 1982(Newman et al, , 1983aCraig and Miller, 1984;Newman and Jasani, 1984;Newman and Hobot, 1987), have some advantages for postembedding staining over epoxy resins. They are hydrophilic and tolerate water during polymerization, hence dehydration of specimens need not be so stringent.…”