A novel, simple, reliable, and sensitive method for multiple immunoenzyme                staining: use of microwave oven heating to block antibody crossreactivity and                retrieve antigens.

Lan, Hui‐Yao; Mu, Wei; Atkins, Robert C.

doi:10.1177/43.1.7822770

Cited by 336 publications

(253 citation statements)

References 8 publications

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“…For antibody detection of p-p38, tissue sections were microwave-treated at 800 W for 12 min in 400 ml of 10 mmol/l sodium citrate, pH 6.0. To prevent antibody cross-reactivity and inactivate previously bound peroxidase-conjugated antibodies [26] in double-immunostaining, p-p38 was always the second antigen detected after microwave treatment. Following immunostaining, some sections were counterstained with periodic acid Schiff's reagent to identify kidney structure and tissue damage.…”

Section: Subjects Materials and Methodsmentioning

confidence: 99%

Abnormal p38 mitogen-activated protein kinase signalling in human and experimental diabetic nephropathy

et al. 2004

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Aims/hypothesis. Inflammation and fibrosis are pathological mechanisms that are partially regulated by cell signalling through the p38 mitogen-activated protein kinase (MAPK) pathway. Elements of the diabetic milieu such as high glucose and advanced glycation endproducts induce activation of this pathway in renal cells. Therefore, we examined whether p38 MAPK signalling is associated with the development of human and experimental diabetic nephropathy. Methods. Immunostaining identified phosphorylated (active) p38 MAPK in human biopsies with no abnormality (n=6) and with Type 2 diabetic nephropathy (n=12). Changes in kidney levels of phosphorylated p38 were assessed by immunostaining and western blotting in mice with streptozotocin-induced Type 1 diabetes that had been killed after 0.5, 2, 3, 4 and 8 months, and in Type 2 diabetic db/db mice at 2, 4, 6 and 8 months of age. Results. Phosphorylated p38 was detected in some intrinsic cells in normal human kidney, including podocytes, cortical tubules and occasional interstitial cells. Greater numbers of these phosphorylated p38+ cells were observed in diabetic patients, and phosphorylated p38 was identified in accumulating interstitial macrophages and myofibroblasts. A similar pattern of p38 activation was observed in both mouse models of diabetes. In mice, kidney levels of phosphorylated p38 increased (2-6 fold) following the onset of Type 1 and Type 2 diabetes. In both mouse models, interstitial phosphorylated p38+ cells were associated with hyperglycaemia, increased HbA 1 c levels and albuminuria. Further assessment of streptozotocin-induced diabetic nephropathy showed that interstitial phosphorylated p38+ cells correlated with interstitial fibrosis (myofibroblasts, collagen). Conclusions/interpretation. Increased p38 MAPK signalling is a feature of human and experimental diabetic nephropathy. Time course studies in mouse models suggest that phosphorylation of p38 plays a pathological role, particularly in the development of interstitial fibrosis.

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Section: Subjects Materials and Methodsmentioning

confidence: 99%

Abnormal p38 mitogen-activated protein kinase signalling in human and experimental diabetic nephropathy

et al. 2004

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“…This labelling procedure was chosen because nuclear antigens, such as Ki-67, are best highlighted in double-imunostaining experiments if developed in a more brilliant black -brown product, whereas cytoplasmic antigens may be easily evidenced also using a lighter red development. Microwave oven heating at 750 W for 5 min was used to block the residual enzyme activity after the first development reaction (Lan et al, 1995). Vascular invasion in NSCLC was highlighted by CD34 immunostaining of endothelial cells using standard immunohistochemical methods.…”

Section: Immunocytochemistry and Evaluation Of Datamentioning

confidence: 99%

Independent prognostic value of fascin immunoreactivity in stage I nonsmall cell lung cancer

et al. 2003

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Fascin-1, the most expressed form of fascin in vertebrate tissues, is an actin-bundling protein that induces cell membrane protrusions and increases motility of normal and transformed epithelial cells. Very few data are available on the role of this protein in nonsmall cell lung cancer (NSCLC). Two hundred and twenty patients with stage I NSCLC and long-term follow-up were evaluated immunocytochemically for fascin expression. Overall, variable fascin immunoreactivity was detected in 98% of 116 squamous cell carcinomas, in 78% of 96 adenocarcinomas, in 83% of six large cell carcinomas, and in the two adenosquamous carcinomas under study. Neoplastic emboli were commonly decorated by the antifascin antibody (Po0.001), also when the surrounding invasive carcinoma was unreactive. Fascin immunoreactivity correlated with high tumour grade (P ¼ 0.017) and, in adenocarcinomas, with high Ki-67 labelling index (P ¼ 0.021). Adenocarcinomas with a prevalent bronchiolo-alveolar in situ component were less commonly immunoreactive for fascin than invasive tumours (P ¼ 0.005). Contralateral thoracic or distant metastases were associated significantly with diffuse (460% immunoreactive tumour cells) fascin expression in adenocarcinomas (P ¼ 0.043), and marginally with strong fascin immunostaining in squamous cell carcinomas (P ¼ 0.13). No associations were noted with any other clinicopathological variables tested. Patients with tumours showing diffuse (460% immunoreactive neoplastic cells) and/or strong immunoreactivity for fascin had a shorter survival (P ¼ 0.006 for adenocarcinomas and P ¼ 0.026 for squamous cell carcinomas), even after multivariate analysis (P ¼ 0.014 and 0.050, respectively). The current study documents for the first time that fascin is upregulated in invasive and more aggressive NSCLC, being an independent prognostic predictor of unfavourable clinical course of the disease. Targetting the fascin pathway could be a novel therapeutic strategy of NSCLC.

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“…After the image of p63 expression was saved using a fluorescent microscope, the section was heated in boiled citrate buffer (0.01 M, pH 6.0) for 10 min to denature bound antibody molecules. This step was necessary to completely block crossreactivity between sequential stainings (Lan et al, 1995), because both primary anti-p63 and anti-TTF1 antibodies used for double staining were raised in mouse. T/ebp staining was carried out as described above, and the expression was visualized with DAB (DAKO).…”

Section: Immunohistochemistrymentioning

confidence: 99%

Origin of the ultimobranchial body cyst: T/ebp/Nkx2.1 expression is required for development and fusion of the ultimobranchial body to the thyroid

Kusakabe

Hoshi

Kimura

2005

Developmental Dynamics

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A novel, simple, reliable, and sensitive method for multiple immunoenzyme staining: use of microwave oven heating to block antibody crossreactivity and retrieve antigens.

Cited by 336 publications

References 8 publications

Abnormal p38 mitogen-activated protein kinase signalling in human and experimental diabetic nephropathy

Abnormal p38 mitogen-activated protein kinase signalling in human and experimental diabetic nephropathy

Independent prognostic value of fascin immunoreactivity in stage I nonsmall cell lung cancer

Origin of the ultimobranchial body cyst: T/ebp/Nkx2.1 expression is required for development and fusion of the ultimobranchial body to the thyroid

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