scholarly journals Immunolocalization of type III collagen in human articular cartilage prepared by high-pressure cryofixation, freeze-substitution, and low-temperature embedding.

1995 ◽  
Vol 43 (4) ◽  
pp. 421-427 ◽  
Author(s):  
R D Young ◽  
P A Lawrence ◽  
V C Duance ◽  
T Aigner ◽  
P Monaghan
Keyword(s):  
High Pressure ◽  
Articular Cartilage ◽  
Polyclonal Antibodies ◽  
Freeze Substitution ◽  
Immunogold Electron Microscopy ◽  
Type Iii Collagen ◽  
Human Articular Cartilage ◽  
Chemical Fixation ◽  
Osteoarthritic Cartilage ◽  
Type Iii

We localized Type III collagen by immunogold electron microscopy in resin sections of intact normal and osteoarthritic human articular cartilage. Comparisons of antibody staining between tissue prepared by high-pressure cryofixation and freeze-substitution without fixatives and that exposed to conventional mild chemical fixation with paraformaldehyde showed that dedicated cryotechniques yielded superior preservation of epitopes that are modified by chemical fixation, and simultaneously provided good ultrastructural preservation. Type III collagen was detected with two polyclonal antibodies, one against the triple-helical domain of the molecule and a second against the more antigenic, globular amino pro-peptide domain, which in this collagen is retained in the extracellular matrix after secretion. Positive labeling was seen in association with the major interstitial fibrils, suggesting co-polymerization of Types III and II collagen in cartilage. Type III collagen could not be detected in aldehyde-fixed normal cartilage. In fixed osteoarthritic cartilage, Type III was detectable only when the antibody to the amino pro-peptide was employed. In contrast, high-pressure cryofixation and freeze-substitution preserved epitopes for both antibodies, permitting immunodetection of Type III collagen in normal and osteoarthritic cartilage. Cryotechniques offer exciting possibilities for significantly improving the immunolocalization of collagens and other fixative-sensitive antigens in situ.

scholarly journals Immunolocalization of Collagen Types II and III in Single Fibrils of Human Articular Cartilage

2000 ◽  
Vol 48 (3) ◽  
pp. 423-432 ◽  
Author(s):  
Robert D. Young ◽  
Paul A. Lawrence ◽  
Victor C. Duance ◽  
Thomas Aigner ◽  
Paul Monaghan
Keyword(s):  
Articular Cartilage ◽  
Polyclonal Antibodies ◽  
Freeze Substitution ◽  
Immunogold Electron Microscopy ◽  
Human Articular Cartilage ◽  
Collagen Types ◽  
Tissue Samples ◽  
Dual Localization ◽  
Fibrillar Collagens ◽  
Secondary Antibodies

Type II and III fibrillar collagens were localized by immunogold electron microscopy in resin sections of human femoral articular cartilage taken from the upper radial zone in specimens from patients with osteoarthritis. Tissue samples stabilized by high-pressure cryofixation were processed by freeze-substitution, either in acetone containing osmium or in methanol without chemical fixatives, before embedding in epoxy or Lowicryl resin, respectively. Ultrastructural preservation was superior with osmium-acetone, although it was not possible to localize collagens by this method. In contrast, in tissue prepared by low-temperature methods without chemical fixation, collagens were successfully localized with mono- or polyclonal antibodies to the helical (Types II and III) and amino-propeptide (Type III procollagen) domains of the molecule. Dual localization using secondary antibodies labeled with 5- or 10-nm gold particles demonstrated the presence of Types II and III collagen associated within single periodic banded fibrils. Collagen fibrils in articular cartilage are understood to be heteropolymers mainly of Types II, IX, and XI collagen. Our observations provide further evidence for the complexity of these assemblies, with the potential for interactions between at least 11 distinct collagen types as well as several non-collagenous components of the extracellular matrix.

Cryo-SEM and TEM of High Pressure Frozen Cells - Some Technical Contributions

2001 ◽  
Vol 7 (S2) ◽  
pp. 728-729
Author(s):  
Paul Walther
Keyword(s):  
High Pressure ◽  
Physiological State ◽  
Electron Microscopic Observation ◽  
Freeze Substitution ◽  
Chemical Fixation ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Electron Microscopic ◽  
Biological Specimen ◽  
Freeze Fracturing

Imaging of fast frozen samples is the most direct approach for electron microscopy of biological specimen in a defined physiological state. It prevents chemical fixation and drying artifacts. High pressure freezing allows for ice-crystal-free cryo-fixation of tissue pieces up to a thickness of 200 urn and a diameter of 2 mm without prefixation. Such a frozen disc, however, is not directly amenable to electron microscopic observation: The structures of interest have to be made amenable to the electron beam, and the structures of interest must produce enough contrast to be recognized in the electron microscope. This can be achieved by freeze fracturing, cryo-sectioning or freeze substitution.The figures show high pressure frozen bakers yeast saccharomyces cerevisiae in the cryo-SEM (Figures 1 and 2) and after freeze substitution in the TEM (Figure 3). For high pressure freezing either a Bal-Tec HPM 010 (Princ. of Liechtenstein; Figures 1 and 2), or a Wohlwend HPF (Wohlwend GmbH, Sennwald, Switzerland; Figure 3) were used.

scholarly journals Ultrastructure of Chondrocytes in Osteoarthritic Femoral Articular Cartilage

2015 ◽  
Vol 11 (3) ◽  
pp. 221-225
Author(s):  
Nj Goya ◽  
M Gupta ◽  
K Joshi
Keyword(s):  
Electron Microscope ◽  
Articular Cartilage ◽  
Control Group ◽  
Human Articular Cartilage ◽  
Joint Motion ◽  
Age Group ◽  
Radiographic Evidence ◽  
Osteoarthritic Cartilage ◽  
Total Knee ◽  
Ultrastructural Findings

Background Osteoarthritis (OA) is a common problem in elderly, but it is not an inevitable feature of ageing. About 80-90% of individuals of both sexes have radiographic evidence of OA by the time they reach an age of 65. But not all of them have the symptoms like pain and decreased joint motion. Objective The objective of the present study was conducted to find out whether the osteoarthritic changes in human articular cartilage are similar to the ageing process or not. Methods Femoral articular cartilage specimens obtained from 13 osteoarthritic patients (52-80years) undergoing total knee replacement and 9 cadavers of same age group (50-80years) (control) were processed and studied under electron microscope. The ultrastructure of the cartilage from the two groups was compared with each other. Results Under the electron microscope, articular cartilage from control group had chondrocytes having a secretary cell characteristic with prominent nucleus and well developed organelles. In osteoarthritic cartilage, degenerating or necrotic chondrocytes were found. Nuclei of these chondrocytes appeared lobulated or indented. Chondrocytes below the fibrillated surface had dilated and irregular endoplasmic reticulum. Electron dense lipid deposits in the extracelluar matrix as well as intracytoplasmic glycogen deposits were much increased in osteoarthritic cartilage as compared to the control group. Amount of perinuclear intracytoplasmic fine filaments was also increased in the chondrocytes of osteoarthritic cartilage. Conclusion Ultrastructural findings of the osteoarthritic articular cartilage were much different from the ageing non-osteoarthritic cartilage. Hence, OA should be considered a specific process and not simply an inevitable feature of ageing. DOI: http://dx.doi.org/10.3126/kumj.v11i3.12507 Kathmandu Univ Med J 2013; 43(3):221-225

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