Usefulness of a quick decalcification of bone sections embedded in methyl metacrylate: an improved method for immunohistochemistry

Gomes, Samirah Abreu; Reis, Luciene M. dos; Oliveira, Ivone B.; Noronha, Irene de Lourdes; Jorgetti, Vanda; Heilberg, Ita Pfeferman

doi:10.1007/s00774-007-0788-2

Cited by 32 publications

(31 citation statements)

References 13 publications

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“…(37) The technique for immunohistochemical detection of bone proteins from clinical samples was adapted from a previously reported method. (39) In brief, two adjacent 5-mm sections of bone tissue were placed side by side on each slide. Bone sections were deacrylated in a 1:1 mixture of xylene and chloroform for 30 minutes, rehydrated in graded alcohol solutions, submitted to a quick decalcification with 1% acetic acid for 10 minutes, and rinsed twice with distilled water.…”

Section: Immunohistochemistry Of Human Bone Biopsiesmentioning

confidence: 99%

Repression of osteocyte Wnt/β-catenin signaling is an early event in the progression of renal osteodystrophy

Sabbagh

Graciolli

O’Brien

et al. 2012

Journal of Bone and Mineral Research

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223

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Chronic kidney disease–mineral bone disorder (CKD‐MBD) is defined by abnormalities in mineral and hormone metabolism, bone histomorphometric changes, and/or the presence of soft‐tissue calcification. Emerging evidence suggests that features of CKD‐MBD may occur early in disease progression and are associated with changes in osteocyte function. To identify early changes in bone, we utilized the jck mouse, a genetic model of polycystic kidney disease that exhibits progressive renal disease. At 6 weeks of age, jck mice have normal renal function and no evidence of bone disease but exhibit continual decline in renal function and death by 20 weeks of age, when approximately 40% to 60% of them have vascular calcification. Temporal changes in serum parameters were identified in jck relative to wild‐type mice from 6 through 18 weeks of age and were subsequently shown to largely mirror serum changes commonly associated with clinical CKD‐MBD. Bone histomorphometry revealed progressive changes associated with increased osteoclast activity and elevated bone formation relative to wild‐type mice. To capture the early molecular and cellular events in the progression of CKD‐MBD we examined cell‐specific pathways associated with bone remodeling at the protein and/or gene expression level. Importantly, a steady increase in the number of cells expressing phosphor‐Ser33/37‐β‐catenin was observed both in mouse and human bones. Overall repression of Wnt/β‐catenin signaling within osteocytes occurred in conjunction with increased expression of Wnt antagonists (SOST and sFRP4) and genes associated with osteoclast activity, including receptor activator of NF‐κB ligand (RANKL). The resulting increase in the RANKL/osteoprotegerin (OPG) ratio correlated with increased osteoclast activity. In late‐stage disease, an apparent repression of genes associated with osteoblast function was observed. These data confirm that jck mice develop progressive biochemical changes in CKD‐MBD and suggest that repression of the Wnt/β‐catenin pathway is involved in the pathogenesis of renal osteodystrophy. © 2012 American Society for Bone and Mineral Research.

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Section: Immunohistochemistry Of Human Bone Biopsiesmentioning

confidence: 99%

Repression of osteocyte Wnt/β-catenin signaling is an early event in the progression of renal osteodystrophy

Sabbagh

Graciolli

O’Brien

et al. 2012

Journal of Bone and Mineral Research

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233

223

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“…Immunostaining for FGF-23, VDR, and Scl was performed in IH patients and controls according to a technique previously described (22). The primary antibodies used were monoclonal anti-human FGF-23 (provided by Susan C. Schiavi, Genzyme, Framingham, MA), rat monoclonal anti-human VDR (ABR-Affinity BioReagents, Inc. Golden, CO), and human monoclonal antibody anti-Scl (R&D Systems, Minneapolis, MN).…”

Section: Immunohistochemistrymentioning

confidence: 99%

Expression of Fibroblast Growth Factor 23, Vitamin D Receptor, and Sclerostin in Bone Tissue from Hypercalciuric Stone Formers

Menon

Moysés

Gomes

et al. 2014

Clinical Journal of the American Society of Nephrology

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Background and objectives Increased bone resorption, low bone formation, and abnormal mineralization have been described in stone formers with idiopathic hypercalciuria. It has been previously shown that the receptor activator of NF-kB ligand mediates bone resorption in idiopathic hypercalciuria (IH). The present study aimed to determine the expression of fibroblast growth factor 23 (FGF-23), vitamin D receptor (VDR), and sclerostin in bone tissue from IH stone formers.Design, setting, participants, & measurements Immunohistochemical analysis was performed in undecalcified bone samples previously obtained for histomorphometry from 30 transiliac bone biopsies of idiopathic hypercalciuria stone-forming patients between 1992 and 2002 and 33 healthy individuals (controls). Serum parameters were obtained from their medical records.Results Histomorphometry disclosed 21 IH patients with high and 9 IH patients with normal bone resorption. Importantly, eroded surfaces (ES/BS) from IH patients but not controls were significantly correlated with VDR immunostaining in osteoblasts (r=0.51; P=0.004), sclerostin immunostaining in osteocytes (r=0.41; P=0.02), and serum 1,25-dihydroxyvitamin D (r=0.55; P,0.01). Of note, both VDR and sclerostin immunostaining were significantly correlated with serum 1,25-dihydroxyvitamin D in IH patients (r=0.52; P=0.01 and r=0.53; P=0.02, respectively), although VDR and sclerostin expression did not differ between IH and controls. IH patients with high bone resorption exhibited a significantly stronger sclerostin immunostaining than IH patients with normal bone resorption. FGF-23 expression in osteocytes from IH patients did not differ from controls and was not correlated with any histomorphometric parameter.Conclusions These findings suggest the contribution of VDR and sclerostin, as well as 1,25-dihydroxyvitamin D, to increase bone resorption in idiopathic hypercalciuria but do not implicate FGF-23 in the bone alterations seen in these patients.

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“…Simultaneous negative controls were carried out by omitting primary antibody in all sections. As positive controls, we used bone sections from patients with CKD-MBD (19). Quantitative expression of OPG, RANKL, and cytokines in bone biopsies were measured by the point-counting technique (23) at a magnification of ϫ100 and using a 176-point ocular grid.…”

Section: Immunohistochemistrymentioning

confidence: 99%

“…Immunostaining for OPG, RANKL, IL-1␣, TGF-␤, and bFGF was performed using the avidin-biotin complex method on undecalcified bone samples that were embedded in methylmetacrylate and submitted to a brief decalcification and pretreatment with Tween 20 for better epitope exposure, as recently described (19). The primary antibodies used were mouse monoclonal anti-human IL-1␣ (R&D Systems, Minneapolis, MN); rabbit polyclonal anti-human TGF-␤ and bFGF (Santa Cruz Biotechnology, Santa Cruz, CA), and goat monoclonal anti-human OPG and RANKL (Santa Cruz Biotechnology).…”

Section: Immunohistochemistrymentioning

confidence: 99%

“…RANKL plays a central role in osteoclast differentiation and can be inactivated by OPG. Recently, the bone expression of OPG and RANKL system was evaluated by means of an effective immunohistochemistry technique that we developed on the basis of a quick decalcification of bone sections embedded in methylmetacrylate (19); however, there have been no data concerning the role of cytokines in bone tissue of patients who have hypercalciuria and present with normal renal function. This study aimed to evaluate the expres-sion of cytokines that are involved in either bone formation or resorption, such as TGF-␤, bFGF, OPG, RANKL, and IL-1␣, in bone biopsy material of IH stone-forming patients.…”

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confidence: 99%

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RANKL Is a Mediator of Bone Resorption in Idiopathic Hypercalciuria

Gomes¹,

Reis²,

Noronha³

et al. 2008

Clinical Journal of the American Society of Nephrology

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Background and objectives: This study aimed to determine the expression of osteoprotegerin, receptor activator of nuclear factor B ligand, interleukin-1␣, transforming growth factor-␤, and basic fibroblast growth factor in stone-forming patients with idiopathic hypercalciuria.Design, setting, participants, & measurements: Immunohistochemical analysis was performed in undecalcified bone samples previously obtained from 36 transiliac bone biopsies of patients who had idiopathic hypercalciuria and whose histomorphometry had shown lower bone volume, increased bone resorption, and prolonged mineralization lag time.Results: Bone expression of receptor activator of nuclear factor B ligand and osteoprotegerin was significantly higher in patients with idiopathic hypercalciuria versus control subjects. Transforming growth factor-␤ immunostaining was lower in patients with idiopathic hypercalciuria than in control subjects and correlated directly with mineralization surface. Interleukin-1␣ and basic fibroblast growth factor staining did not differ between groups. Receptor activator of nuclear factor B ligand bone expression was significantly higher in patients who had idiopathic hypercalciuria and exhibited higher versus normal bone resorption.Conclusion: A higher expression of receptor activator of nuclear factor B ligand in bone tissue suggests that increased bone resorption in patients with idiopathic hypercalciuria is mediated by receptor activator of nuclear factor B ligand. Osteoprotegerin bone expression might have been secondarily increased in an attempt to counteract the actions of receptor activator of nuclear factor B ligand. The low bone expression of transforming growth factor-␤ could contribute to the delayed mineralization found in such patients.

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Usefulness of a quick decalcification of bone sections embedded in methyl metacrylate: an improved method for immunohistochemistry

Cited by 32 publications

References 13 publications

Repression of osteocyte Wnt/β-catenin signaling is an early event in the progression of renal osteodystrophy

Repression of osteocyte Wnt/β-catenin signaling is an early event in the progression of renal osteodystrophy

Expression of Fibroblast Growth Factor 23, Vitamin D Receptor, and Sclerostin in Bone Tissue from Hypercalciuric Stone Formers

RANKL Is a Mediator of Bone Resorption in Idiopathic Hypercalciuria

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