Three important factors are necessary for successful electron microscope autoradiography (EM-ARG): good resolution, proper preparation of the radioactive isotope (RI) labeled diffusible compounds, and shortened exposure time for ARG. The resolution problem is fundamental to EM-ARG. However, unless the diffusible RI compounds have been fixed correctly in the tissues during preparation, good resolution is useless. It is also necessary to shorten the exposure time for ARG. As yet, a high-speed ARG method for electron microscopy has not been reported, although scintillation ARG methods have been applied to macro-and micro-ARG since 1960. High specific activity, a large amount of radioactivity per unit exposure for radio incorporation (incubation), and careful selection of labeled compounds that concentrate in the DNA or RNA of cell organelles may increase the sensitivity of the emulsion and shorten the exposure time for ARG. For example, labeled thymidine accumulates in nuclear DNA, `H-SPG (Schizophyllan-produced polyglucan) is incorporated into lysosomal granules, and labeled iodine concentrates in thyroid follicles, often increasing the sensitivity of the emulsion and shortening the exposure time. High-speed ARG yields good data in a very short time, but high-resolution ARG continues to be necessary, even though it requires 4 weeks or more of exposure time. Scintillation autoradiography using tritium seems unstable. We propose a new way to shorten exposure time for EM-ARG, by combining overdevelopment with coating both sides of the grid with emulsion. This method is approximately 100 times more sensitive than the conventional method, and only 4 days of exposure time are required, in contrast to the 1 month usually needed.
We explored the effects of ovine pituitary-derived renotropin on renal DNA synthesis in castrated hypophysectomized mice. Administration of the preparation at a dose of 47 μg for 5 days was followed by significant increases in renal DNA (134% of controls), and in kidney dry weight, protein and RNA. A time course study showed that [3H]-thymidine incorporation into renal DNA peaked at 8–10 h after one injection (60 μg) 1.9 times higher than in controls. Autoradiographic studies indicated that labeling indices increased significantly in proximal tubules (17 times) and endothelial cells (4 times) in the outer renal medulla of treated mice compared to controls. Nuclear areas in these cells also increased significantly. Our studies demonstrated a time course of new DNA synthesis stimulated by a renotropin and identified renotropin target cells.
"Analytical electron microscope": We have deviced a new analytical electron microscope in Japan for the detection of elemental localization in tissues and cells in 1973.Our analytical EM is basically composed of a JEM 100C transmission electron microscope fitted with a scanning device(ASID-4), a side-entry goniometer stage and an energy dispersive type X-ray microanalyzer unit(EDAX-707B) with an EDIT-Computer system.Freeze substitution method: We compared the results between the freeze dried and the freeze substituted sections of fresh mouse kidney before the cardiac muscle examination. The results were clear that the freeze substitution method with ether or acetone followed by vinylcyclohexane dioxide (ERL-4206) resin mixture seems superior both in the STEM-image and the X-ray microanalysis to that of the freeze dried method.Freshly prepared mouse heart muscle was cut in small pieces within a 3 mm3 in size, mounted on an alminum foil, and frozen with cooled methycyclohexane at the liquid nitrogen temperature, after acrolein vapor fixation for 3 min.
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