Summary
The suitability of four aldehydes (glutaraldehyde, acrolein, formaldehyde, hydroxyadipaldehyde) for histological and histochemical researches has been studied in the following experiments:
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A.
“Small” (0.5–1.0 mm in greatest dimension) and “large” (2.0–5.0 mm) blocks of liver were prefixed in a glutaraldehyde-solution, postfixed in a OsO4-solution and embedded in Epon. In the “small” blocks the fixation was uniformly good. In the “large” blocks it was equally satisfactory in a 0.5 mm thick peripheral zone; deep cell layers however, were not well fixed.
For the fixation by immersion at least two dimensions of a tissue block should not exceed the size of 1 mm.
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B.
Cryostat sections were prepared from “large” blocks of liver after fixation in glutaraldehyde, acrolein, formaldehyde, hydroxyadipaldehyde. On these sections were ascertained the depth of the penetration of the four aldehydes and, after 1 hour washing with distilled water, their speed of fixation with the Schiff reagent. The results shows that the depth of penetration decline from acrolein to formaldehyde, glutaraldehyde and hydroxyadipaldehyde; the speed of fixation from acrolein to glutaraldehyde and formaldehyde. Hydroxyadipaldehyde has practically no fixation effect.
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C.
Mice were stained vitally with neutral red. From their liver were prepared 0.2–0.3 mm thick slices; they were placed between two slides and immersed for 1–2 hs in solutions of the four aldehydes. In the course of the fixation with glutaraldehyde solutions the intensity of the red colour in the peripheral part of the slice increases, the central part on the contrary turn to yellow. We think that some neutral red diffuses from the central part of the slice to its periphery. Both, colour modification and diffusion, are present also with other fixatives containing one (formaldehyde, acrolein, hydroxyadipaldehyde) or two (formaldehyde/glutaraldehyde, acrolein/glutaraldehyde) aldehydes. They are dependant on the type and the concentration of the aldehyde. They could be observed also in “large” tissue blocks prepared from animals receiving neutral red.
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D.
“Large” blocks of the liver prepared from vitally stained mice were prefixed in an aldehyde solution, postfixed in a OsO4-solution and embedded in Epon. The fine structure of the cells in the yellow central zone of these blocks was, after prefixation in glutaraldehyde, worse preserved as in “large” blocks prepared from animals which did not receive neutral red. This is ascribed probably to the large number of autolysosomes which form following the neutral red injection. The fine structure of the cells after prefixation in acrolein is equally satisfactory in the central part as well as in the peripheral.
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E.
In the course of the aldehyde fixation the changes of the pH in “large” blocks were measured with electrodes. The pH generally decreases. The yellow central zone in a block of liver of an animal treated with neutral red has a pH 6.2. Thereafter neutral red is not suitable as pH indicator.
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F.
Spleen and kidney blocks were fixed with glutaraldehyde or hydroxyadipaldehyde solutions. In kryostat sections of the spleen hemoglobin was localized, in those of the kidney the activities of some enzymes (alcaline phosphatase, acid phosphatase, succinic dehydrogenase) were histochemically demonstated. The results show that in the course of the glutaraldehyde fixation hemoglobin diffuses from the central part of the blocks in their periphery. The activity of the alcaline and acid phosphatase are weak in the most peripheral zone of the sections probably because of diffusion of the enzymes in the fixative, or inhibition of the enzyme activities from the fixative, or both. In the adjacent peripheral zone the enzyme activities are well preserved and correctly localized, in the central zone the reaction end products are diffusely arranged. The activity of the succinic dehydrogenase is overall equally localized; it is missing only in a thin peripheral zone of the section which corresponds to the outer layer of the block, where hydroxyadipaldehyde has penetrated.
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Herrn Prof. Dr. med. Helmut Ruska gewidmet.
Durchgeführt mit Unterstützung der Deutschen Forschungsgemeinschaft.
Teile dieser Arbeit wurden in Seminaren (Mailand 1966, Cagliari 1968) und auf der 13. Tagung der Deutschen Gesellschaft für Elektronenmikroskopie (Luciano u. Reale, 1967; Reale u. Luciano, 1967) vorgetragen.
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Reale, E., Luciano, L. Fixierung mit Aldehyden. Ihre Eignungfür histologische und histochemische Untersuchungen in der Licht- und Elektronenmikroskopie. Histochemie 23, 144–170 (1970). https://doi.org/10.1007/BF00305848
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DOI: https://doi.org/10.1007/BF00305848