A novel in situ hybridization signal amplification method based on the                deposition of biotinylated tyramine.

Kerstens, Harold M.J.; Poddighe, Pino J.; Hanselaar, A. G. J. M.

doi:10.1177/43.4.7897179

Cited by 299 publications

(226 citation statements)

References 23 publications

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“…For the second step, the sections were incubated with anti-VEGFR-3 antibodies for 1 hour (0.14 g/ml), followed by biotinylated anti-mouse antibody for 30 minutes (1:200 dilution of the supernatant of clone), ABC-peroxidase for 30 minutes (1:100), biotinylated tyramin solution (1:2.000) containing 0.01% peroxide for 5 minutes, ABC-alkaline phosphatase (1:100) for 20 minutes, and developed with Fast Blue (Sigma) for 20 minutes, according to a procedure previously described for in situ hybridization signal enhancement. 31 Five-m cryosections adjacent to the double stained ones were also immunostained with VEGFR-3 antibodies only, as described above.…”

Section: Vegfr-3 and Pal-e Double Stainingmentioning

confidence: 99%

VEGFR-3 and Its Ligand VEGF-C Are Associated with Angiogenesis in Breast Cancer

Valtola

Salvén

Heikkilä

et al. 1999

The American Journal of Pathology

481

377

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Recently , monoclonal antibodies against the human vascular endothelial growth factor receptor VEGFR-3 were shown to provide a specific antigenic marker for lymphatic endothelium in various normal tissues. In this study we have investigated the expression of VEGFR-3 and its ligand VEGF-C in normal breast tissue and in breast tumors by immunohistochemistry. VEGFR-3 was weakly expressed in capillaries of normal breast tissue and in fibroadenomas. In intraductal breast carcinomas , VEGFR-3 was prominent in the "necklace" vessels adjacent to the basal lamina of the tumor-filled ducts. VEGF receptor 1 and 2 as well as blood vessel endothelial and basal lamina markers were colocalized with VEGFR-3 in many of these vessels. Antibodies against smooth muscle ␣-actin gave a weak staining of the necklace vessels , suggesting that they were incompletely covered by pericytes/smooth muscle cells. A highly elevated number of VEGFR-3 positive vessels was found in invasive breast cancer in comparison with histologically normal breast tissue (P < 0.0001 , the Mann-Whitney test). VEGF-C was located in the cytoplasm of intraductal and invasive cancer cells. The results demonstrate that the expression of VEGFR-3 becomes up-regulated in the endothelium of angiogenic blood vessels in breast cancer. The results also suggest that VEGF-C secreted by the intraductal carcinoma cells acts predominantly as an angiogenic growth factor for blood vessels , although this paracrine signaling network between the cancer cells and the endothelium may also be involved in modifying the permeabilities of both blood and lymphatic vessels and metastasis formation. (Am J Pathol 1999, 154:1381-1390)

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Section: Vegfr-3 and Pal-e Double Stainingmentioning

confidence: 99%

VEGFR-3 and Its Ligand VEGF-C Are Associated with Angiogenesis in Breast Cancer

Valtola

Salvén

Heikkilä

et al. 1999

The American Journal of Pathology

481

377

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“…A modified highly sensitive RNA in situ hybridization method with catalyzed signal amplification based on biotinylated tyramines was employed, which allows messengers to be detected readily with routine formalin-fixed and paraffin-embedded samples. 25 In brief, after hybridization with sense or antisense cRNA hASH1 probe at 501C for 2 1 2 h, the sections were treated with ribonuclease and washed under highly stringent conditions followed by endogenous biotin blocking with a Biotin Blocking System (DAKO, CA, USA). Then, the sections were sequentially incubated with horseradish peroxidase conjugated anti-digoxigenin antibody (DAKO, Denmark), biotinylated tyramide and horseradish peroxidase-labelled streptavidin of the GenPoint kit (DAKO, CA, USA).…”

Section: Tissue Samplesmentioning

confidence: 99%

hASH1 expression is closely correlated with endocrine phenotype and differentiation extent in pulmonary neuroendocrine tumors

et al. 2004

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The human homolog 1 of the Drosophila neurogenic achaete-scute genes, hASH1, is specifically expressed in fetal pulmonary neuroendocrine cells and in some neuroendocrine tumor cell lines. However, no data have been gathered regarding its in vivo expression in tumors. hASH1 mRNA expression was investigated by in situ hybridization in 238 surgically resected lung carcinomas, and the correlations between hASH1 expression status and immunostaining results of neuroendocrine markers chromogranin A, neural cell adhesion molecule, gastrin-releasing peptide and calcitonin, and clinical outcome were analyzed. hASH1 expression was detected in 2/20 (10%) adenocarcinomas, 4/30 (13.3%) typical carcinoids, 11/13 (84.6%) atypical carcinoids, 38/67 (56.7%) large-cell neuroendocrine carcinomas and 56/78 (71.8%) small-cell carcinomas, respectively, but not in any squamous cell carcinoma (0/21) or large-cell carcinoma (0/9). The 2 hASH1 þ adenocarcinomas also expressed multiple neuroendocrine markers. Thus, hASH1 expression was restricted to lung cancers with neuroendocrine phenotypes. However, not all neuroendocrine tumors expressed hASH1. Within the entities of large-cell neuroendocrine carcinoma and small-cell carcinoma, hASH1 expression correlated very closely with chromogranin A, gastrin-releasing peptide and calcitonin expression (Po0.0001, r ¼ 0.852), but was not related to neural cell adhesion molecule expression (P ¼ 0.8892), suggesting that hASH1 expression, at least in lung cancer, is associated with endocrine phenotype expression other than 'neuroendocrine differentiation' in a broad sense. The fact that hASH1 was virtually absent in almost fully differentiated typical carcinoids, but was expressed in most, if not all, less differentiated atypical carcinoids as well as large-cell neuroendocrine carcinomas and small-cell carcinomas, suggests that hASH1 expression in lung cancer imitates its early and transient expression in fetal development, and that hASH1 is instrumental in the establishment, but not in the maintenance, of a cellular endocrine phenotype. Finally, hASH1 expression correlated with a significantly shortened survival in small-cell carcinoma patients (P ¼ 0.041).

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“…The assay kit contains two specific RNA-probes, one for low-risk HPV-types (6,11,42,43,44) and one for high-risk types (16,18,31,33,35, 39, 45, 51, 52, 56, 58, 59, 68), which hybridize with appropriate DNA, gained from a sample collected with a brush from the patient's cervix. The hybrid is bound by polyclonal antibodies which are fixed in the testing tube.…”

Section: Hybrid Capture Testmentioning

confidence: 99%