By Brigitte Schultze
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Extra resources for Autoradiography At the Cellular Level. Physical Techniques in Biological Research
Example text
If specimens are to be stained through the emulsion, the autoradiograms should be allowed to dry completely after photographic processing in order to prevent loss or dislocation of the emulsion during the staining procedure. V. Staining A. STAINING PRIOR TO APPLYING THE EMULSION If the specimens are stained prior to applying the emulsion, they should be slightly overstained, since there will be some loss of the water-soluble dyes during photographic processing. Leblond et al. (1963) and Thurston and Joftes (1963) described some valuable staining procedures that are performed prior to dipping or in combination before and after application of the emulsion.
FREEZE-SUBSTITUTION METHOD A modification of this technique is the freeze-substitution method (Blank et al, 1951; Feder and Sidman, 1958; Russell et al, 1949). Vacuum dehydration is replaced by dehydration in alcohol. Gielink et al (1966) described a method by which the leaching of water-soluble and exchange 45 able calcium ( C a ) in histoautoradiography of oat tissue can be prevented by using acetone as the dehydration fluid and by keeping the tissue sec tions, while stretching on water, embedded in the methacrylate matrix.
There was a loss neither of activity nor of mass of D N A or protein by most of the fixatives examined. For quantitative losses of nucleic acids during the preparation of frozen sections, see Körnender et al. (1965). 2. Differential Fixation As mentioned above, the fixation of protein by fixatives containing protein-precipitating substances has the advantage that the proteins are quantitatively precipitated and the precursor, the free amino acid, is 14 eliminated. Droz and Warshawsky (1963) using leucine- C reported that at least 91-97% of the radioactivity retained in histological sections after application of labeled amino acids is firmly bound to protein, presumably by peptide bonds.