Factors Influencing Metachromatic Staining in Paraffin-Embedded Sections of Rabbit and Human Articular Cartilage: A Comparison of the Safranin O and Toluidine Blue O Techniques

Getzy, Lee L.; Malemud, Charles J.; Goldberg, Victor M.; Moskowitz, Roland W.

doi:10.1179/his.1982.5.3.111

Cited by 36 publications

(12 citation statements)

References 5 publications

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“…One-half of a femoral head and femoral condyle from each animal was fixed in 10% phosphate buffered formalin for 24 hours and processed for paraffin embedding and sectioning after decalcification. Replicate 5 -8~ sections were stained for proteoglycans using Safranin 0, fast green, and iron hematoxylin (16) or toluidine blue 0 (17). Similarly, half of each femoral head was processed for electron microscopy after fixation in 2% paraformaldehyde containing 2.5% glutaraldehyde and 12% picric acid buffered to pH 7.4 with Sorensen's phosphate.…”

Section: Methodsmentioning

confidence: 99%

Metabolic and ultrastructural changes in articular cartilage of rats fed dietary supplements of omega‐3 fatty acids

Lippiello

Fienhold

Grandjean

1990

Arthritis & Rheumatism

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A “marginally deficient” essential fatty acid state was produced in male Sprague‐Dawley rats by dietary supplementation with Ω3 fatty acids. Animals fed diets containing the highest amounts of these fatty acids (10% menhaden fish oil) demonstrated a 70% maximum decrease in the linoleic and arachidonic acid content of articular cartilage, a 30–40% decrease in cartilage hexosamine content, with little effect on hydroxyproline levels, and a 32% inhibition of proteoglycan synthesis. Histologic analysis revealed an occasional surface irregularity and localized depletion of Safranin O and toluidine blue staining of articular cartilage on the femoral heads from animals taking the higher doses. Electron microscopic analysis revealed a marked decrease in “dark‐staining” chondrocytes relative to “light‐staining” cells in all animals fed menhaden fish oil. The cartilaginous changes noted in this study reflect a causal relationship between chondrocyte metabolism and an altered unsaturated fatty acid content. The observed responses of chondrocytes to Ω3 fatty acids may be similar to those commonly associated with the development of early osteoarthrosis. It is not known whether similar changes are induced in other species, including humans, but these observations suggest that some caution must be taken in the long‐term adminitration of menhaden fish oil or other Ω3 fatty acid‐containing preparations in rheumatoid arthritis patients.

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Section: Methodsmentioning

confidence: 99%

Metabolic and ultrastructural changes in articular cartilage of rats fed dietary supplements of omega‐3 fatty acids

Lippiello

Fienhold

Grandjean

1990

Arthritis & Rheumatism

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“…Horizontal slices (2 mm) of selected IVDs devoid of vertebral body and hence requiring no decalcification were fixed directly in 10% neutral buffered formalin for 24 h, dehydrated in graded alcohols, embedded in paraffin wax and sections prepared as outlined above. Following de-paraffinisation in xylene and re-hydration through graded ethanol washes (100-70% v/v) to water, the tissue sections were stained with toluidine blue/ fast green [20] or picro-sirius red [45], the latter stain was examined by polarised light microscopy. The picrosirius red staining method of Sweat et al [45] was used after initial removal of tissue proteoglycans as advocated by Junqueira et al [27] by pre-digestion with bovine testicular hyaluronidase (1,000 U/ml) in 0.1 M phosphate buffer pH 5.0 for 2 h at 37°C.…”

Section: Histologymentioning

confidence: 99%

Aggrecan, versican and type VI collagen are components of annular translamellar crossbridges in the intervertebral disc

et al. 2007

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The aim of this study was to undertake a detailed analysis of the structure of the inter and intralamellar regions of the annulus fibrosus. A total of 30 newborn to 6 year-old lumbar ovine intervertebral discs (IVDs) were fixed and decalcified en-bloc to avoid differential swelling artifacts during processing and vertical midsagittal, and horizontal 4 lm sections were cut. These were stained with toluidine blue to visualise anionic proteoglycan (PG) species, H & E for cellular morphology and picrosirius red (viewed under polarized light) to examine collagenous organization. Immunolocalisations were also undertaken using anti-PG core-protein and glycosaminoglycan (GAG) side chain antibodies to native chondroitin sulphate (CS), D C-4-S and C-6-S unsaturated stubs generated by chondroitinase ABC digestion of CS, keratan sulphate (KS), and with antibodies to type I, II, VI, IX and X collagens. Trans-lamellar cross bridges (TLCBs), discontinuities in annular lamellae's which provide transverse interconnections, stained prominently with toluidine blue in the adult IVDs but less so in the newborn IVDs. In adult discs TLCBs were evident in both the posterior and anterior AF where they extended from the outermost annular lamellae almost to the transitional zone extending across as many as eight lamellar layers displaying a characteristic circuitous, meandering, serpentine type course. There were significantly fewer TLCBs in 2 week-old compared with skeletally mature sheep and there was a further increase from 2 to 6 years. Immunolocalisation of perlecan delineated blood vessels in the TLBs of the newborn but not adult IVDs extending into the mid AF. In contrast adult but not 2 week-old TLCBs were immunpositive for C-4-S, C-6-S, KS, aggrecan, versican and type VI collagen. The change in number and matrix components of the translamellar cross bridges with skeletal maturity and ageing suggest that they represent an adaptation to the complex biomechanical forces occurring in the annulus fibrosus.

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“…Neutral formalin-fixed tissue sections (4 µm) were routinely stained for tissue PGs using a modification of the method of Getzy et al (1982). Tissue sections were stained in 0.04% (w/v) toluidine blue O in 100 mM sodium acetate pH 4.0 (10 min), briefly rinsed in distilled water, counterstained 2-3 min in 0.1% (w/v) aqueous fast green FCF and then rinsed again in distilled water.…”

Section: Histochemistrymentioning

confidence: 99%

Perlecan, the multidomain HS-proteoglycan of basement membranes, is a prominent pericellular component of ovine hypertrophic vertebral growth plate and cartilaginous endplate chondrocytes

Melrose

Smith

Knox

et al. 2002

Histochem Cell Biol

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The aim of this study was to immunolocalise perlecan in ovine vertebral growth plate (VGP) and cartilaginous endplate (CEP) cartilages using a monoclonal antibody (MAb A76) directed to a core protein epitope in perlecan domain-I, and to compare and contrast its localisation patterns with known cartilage matrix components. Perlecan was a prominent pericellular component of mature hypertrophic chondrocytes in the VGP and CEP in newborn 2-to 5-day-old sheep. Type I, II, VI and X collagen, chondroitin-4 and 6-sulphate, 7-D-4 chondroitin sulphate isomer proteoglycan epitope, keratan sulphate, aggrecan core protein, hyaluronan (HA) and hyaluronan binding proteins (HABPs) each had distinct localisation patterns in the VGP and CEP. Type X collagen was a prominent component of the VGP but was undetectable in the CEP. Aggrecan was strongly localised extracellularly throughout the VGP and CEP but increased cellassociated staining was also evident. In contrast to the aforementioned matrix components, HA, HABPs and perlecan were localised strongly to the pericellular matrices of the hypertrophic VGP and CEP chondrocytes apparently indicating an important role for these components in terminal chondrocyte differentiation.Perlecan, the multidomain HS-proteoglycan of basement membranes, is a prominent pericellular component of ovine hypertrophic vertebral growth plate and cartilaginous endplate chondrocytes

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Factors Influencing Metachromatic Staining in Paraffin-Embedded Sections of Rabbit and Human Articular Cartilage: A Comparison of the Safranin O and Toluidine Blue O Techniques

Cited by 36 publications

References 5 publications

Metabolic and ultrastructural changes in articular cartilage of rats fed dietary supplements of omega‐3 fatty acids

Metabolic and ultrastructural changes in articular cartilage of rats fed dietary supplements of omega‐3 fatty acids

Aggrecan, versican and type VI collagen are components of annular translamellar crossbridges in the intervertebral disc

Perlecan, the multidomain HS-proteoglycan of basement membranes, is a prominent pericellular component of ovine hypertrophic vertebral growth plate and cartilaginous endplate chondrocytes

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