Antibody penetration into LR-White sections

Brorson, Sverre‐Henning; Roos, Norbert; rten, Fredrik Skj

doi:10.1016/0968-4328(94)00035-2

Cited by 32 publications

(12 citation statements)

References 13 publications

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“…Immunogold labelling with antibodies against BSP and OPN was performed as previously described [32]. Micrographs were obtained by systematic random sampling of cells/surrounding matrix and analyzed using the semiautomatic interactive image analyzer software AnalySIS® pro (Soft Imaging System, Münster, Germany).…”

Section: Methodsmentioning

confidence: 99%

The Skeletal Phenotype of Chondroadherin Deficient Mice

et al. 2013

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Chondroadherin, a leucine rich repeat extracellular matrix protein with functions in cell to matrix interactions, binds cells via their α2β1 integrin as well as via cell surface proteoglycans, providing for different sets of signals to the cell. Additionally, the protein acts as an anchor to the matrix by binding tightly to collagens type I and II as well as type VI. We generated mice with inactivated chondroadherin gene to provide integrated studies of the role of the protein. The null mice presented distinct phenotypes with affected cartilage as well as bone. At 3–6 weeks of age the epiphyseal growth plate was widened most pronounced in the proliferative zone. The proteome of the femoral head articular cartilage at 4 months of age showed some distinct differences, with increased deposition of cartilage intermediate layer protein 1 and fibronectin in the chondroadherin deficient mice, more pronounced in the female. Other proteins show decreased levels in the deficient mice, particularly pronounced for matrilin-1, thrombospondin-1 and notably the members of the α1-antitrypsin family of proteinase inhibitors as well as for a member of the bone morphogenetic protein growth factor family. Thus, cartilage homeostasis is distinctly altered. The bone phenotype was expressed in several ways. The number of bone sialoprotein mRNA expressing cells in the proximal tibial metaphysic was decreased and the osteoid surface was increased possibly indicating a change in mineral metabolism. Micro-CT revealed lower cortical thickness and increased structure model index, i.e. the amount of plates and rods composing the bone trabeculas. The structural changes were paralleled by loss of function, where the null mice showed lower femoral neck failure load and tibial strength during mechanical testing at 4 months of age. The skeletal phenotype points at a role for chondroadherin in both bone and cartilage homeostasis, however, without leading to altered longitudinal growth.

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Section: Methodsmentioning

confidence: 99%

The Skeletal Phenotype of Chondroadherin Deficient Mice

et al. 2013

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“…Immunogold labeling was performed as earlier described [34]. Nonspecific rabbit IgG served as a negative control.…”

Section: Methodsmentioning

confidence: 99%

Increased Tartrate-Resistant Acid Phosphatase Expression in Osteoblasts and Osteocytes in Experimental Osteoporosis in Rats

et al. 2014

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Tartrate-resistant acid phosphatase (TRAP) is known as an osteoclast marker, but osteoblasts and osteocytes in the vicinity of bone remodeling sites also express TRAP. Cell culture studies suggest that osteoblasts endocytose osteoclastic TRAP for inactivation. To evaluate whether changes in osteoclast activity could alter TRAP expression in osteoblasts and/or osteocytes in vivo, we studied the ovariectomized and vitamin D-deficient rat (Ovx-D) and rats healing from rickets. Bone sections were analyzed for TRAP gene expression by in situ hybridization, TRAP protein by immunogold labeling, and TRAP enzyme activity using the fluorescent substrate ELF97. Osteoblasts and osteocytes close to intracortical remodeling sites and bone surfaces demonstrated TRAP, most prominently in cancellous bone and osteocytes. Intracellular TRAP was located to electron-dense vesicles with similar morphology in both cell types. Ovx-D increased osteoclast activity (p < 0.001) and ELF97+ osteocytes (p < 0.05) in cancellous bone, but no corresponding increase was observed in the osteocyte lacunar area. The level of TRAP+ vesicles in cortical osteoblasts (p < 0.01) in Ovx-D rats was also increased. Enhanced osteoclast activity was noted in healing rickets after 72 h (p < 0.05), but no differences in TRAP expression were detected in osteoblasts or osteocytes. Thus, increased osteoclast activity does not affect TRAP expression in osteoblasts and osteocytes, favoring the notion that increased TRAP in these cells is rather due to increased synthesis. Although the role of TRAP in osteoblasts and osteocytes remains elusive, we speculate that the function is related to the capability of the enzyme to regulate the phosphorylation of proteins known to be expressed by these cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s00223-013-9834-3) contains supplementary material, which is available to authorized users.

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“…Immunogold labeling was done on grids for all LR White sections, whether they had been exposed to sodium ethoxide or not (Brorson et al 1994). For the primary antibodies, the incubation was carried out overnight at 4C (fibrin clots, anti-fibrinogen; thyroid, anti-thyroglobulin; kidney, anti-IgA, anti-IgG, anti-IgM), and for the secondary immunoreagents the conditions of incubation were 1 hr at 22C.…”

Section: Methodsmentioning

confidence: 99%

“…Kidney tissue was obtained from a needle biopsy taken at Ullevål Hospital. Fibrin clots were prepared by a method described earlier (Brorson et al 1994).…”

Section: Methodsmentioning

confidence: 99%

How to Examine the Antigen-damaging Effect of Sodium Ethoxide on Deplasticized Epoxy Sections

Brorson

1997

J Histochem Cytochem.

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The purpose of this investigation was to develop a method that could be used to estimate how damaging sodium ethoxide is to different antigens with respect to immunolabeling when epoxy sections are deplasticized. If we obtain weak labeling for an antigen on deplasticized epoxy sections, this might be caused by the damaging effect of the ethoxide solution. It is therefore interesting to develop a method to check if this really is the reason. Fibrin clots and tissues of human kidney and thyroid were embedded in LR White resin. Some thin sections from these specimen blocks were exposed to sodium ethoxide in the same way as epoxy sections are when being deplasticized. Other sections from the same blocks were not exposed to sodium ethoxide. Both categories of sections were immunogold-labeled with anti-fibrinogen, anti-thyroglobulin, anti-IgA, anti-IgG, or anti-IgM. The intensity of immunolabeling of sections treated with ethoxide was compared with the immunolabeling of corresponding sections that were not treated with ethoxide. No significant differences were found in immunolabeling for fibrinogen, IgA, IgG, and IgM. For thyroglobulin, the intensity was approximately 30% less in tissues that were exposed to sodium ethoxide. The practical significance of this method is that we easily can examine the degree to which a given antigen is affected by sodium ethoxide, which is the agent used for deplasticizing epoxy sections.

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Antibody penetration into LR-White sections

Cited by 32 publications

References 13 publications

The Skeletal Phenotype of Chondroadherin Deficient Mice

The Skeletal Phenotype of Chondroadherin Deficient Mice

Increased Tartrate-Resistant Acid Phosphatase Expression in Osteoblasts and Osteocytes in Experimental Osteoporosis in Rats

How to Examine the Antigen-damaging Effect of Sodium Ethoxide on Deplasticized Epoxy Sections

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