Techniques of radioautography for medical and biological research

Abstract

Standard techniques for radioautography used in biological and medical research can be classified into three categories, i.e., macroscopic radioautography, light microscopic radioautography and electron microscopic radioautography. The routine techniques used in these three procedures are described. With regard to macroscopic radioautography, whole body radioautography is a standard technique which employs freezing and cryosectioning and can demonstrate organ distributions of both soluble and insoluble compounds. In contrast, in light and electron microscopic radioautography, soluble and insoluble techniques are separated. In order to demonstrate insoluble labeled compounds, conventional chemical fixations such as formalin for light microscopy or buffered glutaraldehyde and osmium tetroxide for both light and electron microscopy followed by dehydration, embedding and wet-mounting applications of radioautographic emulsions can be used. For the demonstration of soluble labeled compounds, however, cryotechniques such as cryofixation, cryosectioning, freezedrying, freeze-substitution followed by dry-sectioning and dry-mounting radioautography should be employed both for light and electron microscopy. The outlines of these techniques, which should be utilized in various fields of biological and medical research, are described in detail.

Macromolecular Synthesis in the Digestive and Respiratory Systems

“…The results obtained from the tissues of 3 groups of animals injected respectively with 3 kinds of RI-labeled precursors, i.e. 3 H-thymidine, 3 H-uridine and 3 H-leucine, were already reported as several original articles and reviews (Ma 1988,b, 1990a,b, 2000, Ma et al 1991, Nagata 1996a,b, 1997a, 1998a, 1999c, 2001c, 2002, 2003, 2006a,b, 2007a,b, 2009a or as a monograph in the series of Prog. Histochem.…”

Three-Dimensional and Four-Dimensional Observation of Histochemical and Cytochemical Specimens by High Voltage Electron Microscopy.

“…For radioautography, various organs of mice and rats were used after injection of radiolabeled compounds. To observe the intracellular localization of macromolecular synthesis, radiolabeled macromolecular precursors such as 3 H-thymidine for DNA synthesis, 3 H-uridine for RNA synthesis, 3 H-amino acids for protein synthesis, 3 H-glucosamine and 35 SO4 for glucide synthesis, 3 H-fatty acids and 3 H-glycerol for lipid synthesis were used [27,29,30]. Other radiolabeled compounds which bind to the specific cell organelles can also be used.…”

“…Other radiolabeled compounds which bind to the specific cell organelles can also be used. The tissues were taken from the animals injected intraperitoneally with radiolabeled compounds 1 hr before sacrifice and the tissues were doubly fixed in 0.2% glutaraldehyde solution in cacodylate buffer (pH 7.4) and 1.0% osmium tetroxide solution in the same buffer, embedded in Epok 812 resin, sectioned at 0.2 mm thick, and processed for radioautography [20,30].…”

“…To observe the intracellular localization of macromolecular synthesis, radiolabeled macromolecular precursors such as 3 H-thymidine, 3 [5,27,29,30,35]. Other radiolabeled compounds which bind to the specific cell organelles can also be used.…”

“…The liver and pancreatic tissues were taken out, fixed doubly in 2.5% glutaraldehyde solution in cacodylate buffer (pH 7.4) and 1.0% osmium tetroxide solution in the same buffer, dehydrated, embedded in Epok 812 resin, cut at 0.2 mm thick sections and picked up onto collodion coated copper EM grids. For in vitro labeling, cells were plated in 35 mm plastic Petri dishes containing formval and collagen coated gold grid meshes, cultured in a medium containing radioactive compounds, such as 3 H-thymidine, 3 H-uridine (Amersham, England, specific activity 877GBq/mM, 37-1850 KBq/ml medium), 3 Hamino acids, 3 H-glucosamine and 35 SO4, 3 H-fatty acids and 3 H-glycerol, which are precursors for macromolecular synthesis [5,27,29,30,35] or 14 C-bezafibrate and clofibrate (Boehringer Mannheim GmbH, Germany, specific activity 2.83 MBq/mg, concentration 3.7 MBq/ml medium), which are peroxisome proliferators and bind to endoplasmic reticulum and peroxisomes [20], for times varying from 10 min to 60 min. After the labeling, cells were fixed in 2% paraformaldehyde solution in phosphate buffer at pH 7.4 for 1 hr, postfixed in 1% osmium tetroxide solution for 1 hr in the same buffer according to the conventional chemical fixation to demonstrate insoluble compounds [27,29,30].…”