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The osmium tetroxide-p-phenylenediamine procedure reveals the chromatid cores and kinetochores of meiotic chromosomes by light and electron microscopy
We analyzed first-metaphase meiotic chromosomes of the grasshopper Chorthippus jucundus by two different methods, i.e., a silver impregnation technique and the osmium tetroxide-p-phenylenediamine (Os-PPD) procedure. The former was applied on squashed testes previously fixed in ethanol-acetic acid, w...
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Published in: | The journal of histochemistry and cytochemistry 1996-11, Vol.44 (11), p.1279-1288 |
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Main Authors: | , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that cite this one |
Online Access: | Get full text |
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Summary: | We analyzed first-metaphase meiotic chromosomes of the grasshopper Chorthippus
jucundus by two different methods, i.e., a silver impregnation technique and the
osmium tetroxide-p-phenylenediamine (Os-PPD) procedure. The former was applied on
squashed testes previously fixed in ethanol-acetic acid, whereas for Os-PPD the
material was not subjected to any previous extraction treatment but was fixed in
OsO4, treated with PPD, and embedded in Epon 812. Both techniques revealed chromatid
cores and kinetochores regardless of the processing of the material (squashed or
sectioned). Unstained Os-PPD sections were analyzed by light microscopy and
transmission electron microscopy (TEM). The Os-PPD technique provided a high contrast
of chromatid cores and kinetochores in relation to the chromatin, which revealed a
low electron density. To determine the Os-PPD reaction mechanism, the PAS procedure,
as well as scanning electron microscopy (SEM) backscattering and SEM X-ray
microanalysis, was performed on sections. By use of the Os-PPD-PAS procedure, glycol
groups formed by oxidation of osmium bound to aromatic substrates were detected in
chromatid cores and kinetochores by brightfield and fluorescence microscopy. A high Z
contrast was detected in these structures by backscattered electron imaging. SEM
X-ray microanalysis showed osmium and phosphorus to be the main elements present on
the chromatid cores. Taking into account the known reactivity of OsO4 and the present
results, the possible participation of nucleic acids as well as proteins in the
Os-PPD reaction mechanism and in the composition of chromatid cores and kinetochores
is discussed. |
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ISSN: | 0022-1554 1551-5044 |
DOI: | 10.1177/44.11.8918903 |