A staining procedure for melanin in semithin and ultrathin epoxy section
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Abstract:
Staining for melanin at the ultrastructural level may act as an important diagnostic aid by helping to identify apparently amelanotic melanomas. A modified Warthin‐Starry (WS) procedure for melanin has therefore been adapted for direct application to epoxy sections. Minute amounts of melanin can thus be demonstrated in individual melanosomes, indicating a high sensitivity. Using the usual types of fixation, specificity for melanin at the ultrastructural level is high. Primary osmium tetroxide (OsO 4 ) fixation probably induces false positive staining of lipofuscin and should not be used prior to the WS procedure, but it does not influence the results of the procedure when employed as a post‐fixative. It is not improbable, however, that the positive staining of lipofuscin and also of mast cell granules is due to the presence of melanin in these structures. The WS procedure yields additional diagnostic information, especially in suboptimally preserved material, as expressed by an increase in the number of convincingly identifiable melanosomes in one completely and four partly amelanotic melanomas.Keywords:
Melanosome
Osmium tetroxide
Fixative
Lipofuscin
A method is described for the sequential fixation of cell suspensions, suitable for use at room or culture temperatures. Though an adequate method for fixing cell suspensions does exist in the literature (Hirsch & Fedorko, 1968), it involves the use of a mixed glutaraldehyde-osmium tetroxide fixative. Since these two components inter-react, this method has many drawbacks. Previously described weaknesses of a sequential fixation regime (Hirsch & Fedorko, 1968; Jones, Yeh & Hirsch, 1972) with glutaraldehyde and osmium tetroxide have been overcome by the use of vacuum distilled glutaraldehyde as the primary fixative. The results, using a mixed glutaraldehyde-osmium tetroxide fixative and using the two components sequentially on a variety of cell types, are compared. The advantages of a sequential fixation made possible by the use of vacuum distilled glutaraldehyde rather than commercial glutaraldehyde are discussed.
Glutaraldehyde
Fixative
Osmium tetroxide
Distilled water
Osmium
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The retinal pigment epithelium contains three major types of pigment granules; melanosomes, lipofuscin and melanolipofuscin. Melanosomes in the retinal pigment epithelium (RPE) are formed during embryogenesis and mature during early postnatal life while lipofuscin and melanolipofuscin granules accumulate as a function of age. The difficulty in studying the formation and consequences of melanosomes and lipofuscin granules in RPE cell culture is compounded by the fact that these pigment granules do not normally occur in established RPE cell lines and pigment granules are rapidly lost in adult human primary culture. This review will consider options available for overcoming these limitations and permitting the study of melanosomes and lipofuscin in cell culture and will briefly evaluate the advantages and disadvantages of the different protocols.
Lipofuscin
Melanosome
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SUMMARY Cell bodies of cerebral neurons from rabbits were isolated by hand, transferred to a microscope slide in a ‘199’ medium, and the projected areas of their cytoplasm and nuclei were measured. In sixty‐four cells there was a strong correlation between the projected areas of the cytoplasm and the nuclei ( r =0.66, P < 0001), and the ratio of the projected areas was 11.6. The medium was then replaced by the following fixatives: formalin (10% v/v), Bouin's, Carnoy's, Susas, glutaraldehyde (5% v/v), and osmium tetroxide (1% w/v). Cerebral slices were obtained from the grey and white matter of rabbit and rat, and were also measured before and after treatment with similar fixatives. Relative to the unfixed areas, glutaraldehyde and osmium tetroxide had no significant effect on the projected areas of isolated cells, Carnoy's fixative shrunk the areas of the cytoplasm by means of 16.26%, Bouin's by a mean of 49%, and Susas by a mean of 65%. The shrinkage of the cytoplasm and the nuclei was not significantly different from that of the nuclei for each of these three fixatives individually, but with formalin the mean shrinkage of the cytoplasm was 46% while the nuclei did not shrink significantly. Using the same fixatives the effect on the areas of the cerebral slides from rabbit and rat were as follows: glutaraldehyde and osmium tetroxide caused no change in area; Carnoy's, formalin and Bouin's fixative diminished the areas by a mean of 10–20%, and Susa's by a mean of 35%. It was concluded that a particular fixative often caused a different degree of shrinkage to the cytoplasm, nuclei and cerebral slice. In general, the lower the osmotic pressure of the fixative, the less shrinkage it induced.
Fixative
Osmium tetroxide
Glutaraldehyde
Rabbit (cipher)
Osmium
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Lipofuscin
Melanosome
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Citations (1)
Fixative
Osmium tetroxide
Glutaraldehyde
Osmotic concentration
Osmium
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Melanosome
Melanocyte
Granule (geology)
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This investigation was carried out in order to find out which was the best technique, among those reported in the literature, to recognize the small lymph vessels by light microscopy, while at the same time allowing a good ultrastructural preservation of tissues. The study was performed with rabbit hearts according to the following methods: (1) injection of India ink into the wall of the heart followed by perfusion with aldehyde fixative; (2) injection of India ink into the wall of the heart followed by immersion in aldehyde fixative; (3) treatment of the animals with histamine dihydrochloride by e.v. route and aldehyde (4) fixation by immersion; (5) fixation by immersion in osmium tetroxide fixative; fixation by immersion in aldehyde fixative; (6) fixation by perfusion with the same mixture as in (5). In cases (1), (2), (3), (5), and (6), the tissue specimens were postfixed in osmium tetroxide according to conventional methods. The validity of the different methods was checked by examining semithin sections of varying thickness at the light microscope and ultrathin sections at the transmission electron microscope. A comparative examination of the preparation showed that the method allowing the promptest recognition of the small lymph vessels, together with the best ultrastructural preservation and a good reproducibility of results was perfusion of the coronary circle.
Fixative
Osmium tetroxide
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Fixative
Osmium tetroxide
Glutaraldehyde
Osmotic concentration
Osmium
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Citations (87)
Melanosome
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Fixative
Osmium tetroxide
Glutaraldehyde
Microanalysis
Paraformaldehyde
Osmium
Cytochemistry
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