The efficiency of immunolabel on Lowicryl sections compared to theoretical predictions.

Abstract

The surface of thin sections of aldehyde-fixed biological material shows a specimen-related relief of 2-6 nm with Lowicryl. Epon sections are about three times smoother. The relief is the consequence of thin-sectioning being in reality a cleavage. Epitopes are supposed to be laid open (or set free) because cleavage follows the interfaces between protein and Lowicryl. We have developed a simple theory on this basis and have theoretically estimated the efficiency of on-section labeling and compared it with experimental data. For randomly dispersed proteins in cytoplasm, Lowicryl sections will yield significant label only when the concentration of the antigen is about 10 microM or more. The complex situation of more compact proteins, as represented by fibers, sheets, and biological membranes is discussed and the difficulty of significant calculations is explained. Pre-embedding labeling and melted cryosections should give 10-30 times more label. The possible reasons for the observed much smaller gain of not more than two to three times are discussed.

Correlated Atomic Force and Transmission Electron Microscopy of Nanotubular Structures in Pulmonary Surfactant

“…Previous studies have suggested that these cross-hatch structures may be surfactant protein A embedded in the lipid-tubular core (Beckmann and Dierichs, 1984). The surface textures of TM in the Lowicryl sections seen here are in agreement with previous studies by scanning electron microscopy of bacterial sections in which specimen-related reliefs of 5-6 nm were found near membranous organelles (Kellenberger et al, 1987). Some of the surface texture of the larger areas of the section observed by AFM at low magnification corresponds to previously published images of sections of cellular regions embedded in different polymeric resins observed using AFM (Yamamoto and Tashiro, 1994;Braet et al, 1997).…”

“…Some studies have suggested that biological tissue sections used for TEM possess different degrees of surface roughness, depending on the polymeric materials used (Yamamoto and Tashiro, 1994). Others have shown that due to the lack of copolymerization of some biological materials with embedding materials, such as Lowicryl, the cleavage (or sectioning) surface follows areas of least resistance and epitopes are laid open in such surface sections (Kellenberger et al, 1987). The path of least resistance during cutting or sectioning of polymerembedded cell organelles is provided normally by the interfaces between the polymeric resins and proteins (Kellenberger et al, 1987).…”

“…Others have shown that due to the lack of copolymerization of some biological materials with embedding materials, such as Lowicryl, the cleavage (or sectioning) surface follows areas of least resistance and epitopes are laid open in such surface sections (Kellenberger et al, 1987). The path of least resistance during cutting or sectioning of polymerembedded cell organelles is provided normally by the interfaces between the polymeric resins and proteins (Kellenberger et al, 1987). The reason this process occurs during cutting is that the tissues are embedded at low temperature and cleaved at higher ambient temperature.…”

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.…”

Method for fabrication and verification of conjugated nanoparticle-antibody tuning elements for multiplexed electrochemical biosensors

“…Although protein-conjugated gold nanoparticles have been successfully employed as biological markers for decades, instances of total loss (Horrisberger, 1977) or partial loss of specific activity of a protein upon conjugation have also been reported (Kellenberger, 1987;Park, 1986). The study by Bauer et.…”