Freeze-substitution staining of rat growth plate cartilage with alcian blue for electron microscopic study of proteoglycans

To selectively stain polyanionic macromolecules of growth plate cartilage and to prevent artifacts induced by aqueous fixation, proximal tibial growth plates were excised from rats, slam-frozen, and freeze-substituted in 100% methanol containing the cationic dye Alcian blue. Electron microscopic exa...

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Bibliographic Details
Published in:The journal of histochemistry and cytochemistry 1989-03, Vol.37 (3), p.383-387
Main Authors: Maitland, ME, Arsenault, AL
Format: Article
Language:English
Subjects:
Alcian Blue
Animals
Biological and medical sciences
Copper - analysis
Diverse techniques
Electron Probe Microanalysis
Extracellular Matrix - analysis
Freezing
Fundamental and applied biological sciences. Psychology
Growth Plate - analysis
Growth Plate - ultrastructure
Indoles
Microscopy, Electron
Molecular and cellular biology
Proteoglycans - analysis
Rats
Rats, Inbred Strains
Staining and Labeling - methods
Sulfur - analysis
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Summary:To selectively stain polyanionic macromolecules of growth plate cartilage and to prevent artifacts induced by aqueous fixation, proximal tibial growth plates were excised from rats, slam-frozen, and freeze-substituted in 100% methanol containing the cationic dye Alcian blue. Electron microscopic examination showed the tissue stained with Alcian blue to be comparable in ultrastructural preservation to tissues slam-frozen and freeze-substituted in the absence of Alcian blue. The extracellular matrix exhibited a characteristic staining pattern when stained by this method. The pericellular rim was identified as a band of varying width encircling the chondrocyte and its cell processes. Peripheral to the pericellular rim the heterogeneity of staining within the extracellular matrix increased, taking the form of polymorphic densities. X-ray microanalysis showed that the visual interpretation of electron density was related to the concentration of copper present, and that the concentration of sulfur was variable in the pericellular rim and in the interterritorial matrix. The difficulties associated with aqueous fixation and staining procedures are discussed in contrast to the improved preservation achieved by cryogenic methods.
ISSN:0022-1554
1551-5044
DOI:10.1177/37.3.2465336