Quantitative analysis of chromosome in situ hybridization signal in paraffin‐embedded tissue sections

Dhingra, Kapil; Sneige, Nour; Pandita, Tej K.; Johnston, Dennis A.; Lee, J. S.; Emami, Kamal; Hortobágyi, Gabriel N.; Hittelman, Walter N.

doi:10.1002/cyto.990160203

Cited by 36 publications

(29 citation statements)

References 19 publications

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“…[21][22][23] In general, quantitative analysis of thin sections is compromised by the fact that most cells are not wholly intact in thin sections, and the error becomes greater when the object size is larger relative to section thickness. In this study, we initially detected EGFP þ F-actin þ tubular epithelial cells in 6-mm cryostat sections, as this thickness was within the range of those used in other studies (commonly 4-8 mm thickness sections).…”

Section: Discussionmentioning

confidence: 99%

3D-confocal structural analysis of bone marrow-derived renal tubular cells during renal ischemia/reperfusion injury

Toyokawa

Nakao

Stolz

et al. 2006

Laboratory Investigation

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Bone marrow cells (BMC) have been shown to migrate into injured sites for parenchymal repair. However, the extent of BMC involvement is controversial. To determine whether and to what extent BMC contribute to renal parenchymal repair, we employed three-dimensional (3D) fluorescent confocal microscopy/video in renal warm and cold ischemia/reperfusion (I/R) injury using enhanced green fluorescent protein transgenic rats and their radiation chimeras. After induction of renal warm I/R injury in chimeras, BM-derived renal tubular cells were found in 2D microscopy as isolated single cells or clusters of 2-3 cells. Likewise, cold I/R injury resulted in hostderived tubular cells with frequencies B0.2%. However, stringent confocal microscopic analysis and 3D image construction revealed that BM-derived tubules identified in 2D images were frequently artifacts of overlapping cells separately stained with different markers. The actual frequency in 3D analysis was approximately onefourth of that seen in 2D analysis. 3D confocal imaging precisely detected BM-derived tubular epithelial cells and could be useful to study BMC contribution to tissue repair. Although there are abundant data on the phenotype, isolation, culturing, expansion, and differentiation potential of stem cells in the bone marrow, their role and functional relevance in normal adult life or regenerative parenchymal cell turnover is unclear. In addition, many of the findings in this new field are controversial, partly due to the inherent difficulties of techniques used to assess the plasticity of these cells.In this study, transgenic rats carrying the enhanced green fluorescent protein (EGFP) transgene were used to demonstrate three-dimensional (3D) structure analysis of the differentiation of BMC to renal tubular epithelial cells after renal ischemia/ reperfusion (I/R) injury. The results suggest that BMderived cells can differentiate into renal tubular epithelial cells after I/R injury; however, the frequency is considerably less with 3D analysis than that identified with conventional 2D microscopic analyses of thin sections. Materials and methods AnimalsEGFP-transgenic and WT Sprague-Dawley (SD) rats, originally generated by Dr Masaru Okabe (University

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Section: Discussionmentioning

confidence: 99%

3D-confocal structural analysis of bone marrow-derived renal tubular cells during renal ischemia/reperfusion injury

Toyokawa

Nakao

Stolz

et al. 2006

Laboratory Investigation

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“…The tissue showed the same morphology, whereas the cytogelatter findings contradict the statement that, due to trun-netic status appeared different. As shown above, loss of cation effects, monosomy in a significant proportion of information concerning focal cytogenetic alterations is cells will be easily missed (Dhingra et al 1994). …”

Section: Discussionmentioning

confidence: 99%

Interphase in situ hybridization to disaggregated and intact tissue specimens of prostatic adenocarcinomas

Alers

Krijtenburg

Vissers

et al. 1995

Histochem Cell Biol

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A comparative study was performed of interphase in situ hybridization (ISH) to deparaffinized 4-gm tissue sections and nuclear suspensions from eight prostatic adenocarcinomas, as well as one normal prostatic control. Whole nuclear suspensions were derived from the same tumor areas to evaluate differences of ISH to truncated versus whole nuclei. DNA probes specific for the centromeres of chromosome l, 7, 8, 10, and Y were used for detection of numerical chromosomal changes and aneuploidy. In six adenocarcinomas chromosome aberrations (+7, +8, -8, -10, -Y) were seen. However, ISH to sections revealed focal aberrations (-10, -Y) in four cases that could not be distinguished in the suspensions. Chromosomal alterations occurring in larger tumor areas were also detected in the nuclear suspensions. Chromosome copy number changes, especially gains, were better discriminated in the nuclear suspensions. The rate of ISH aneuploidy seen in nuclear suspensions corresponded with that observed in the tissue sections (P<0.01). Ploidy patterns as assessed by ISH to sections and nuclear suspensions were in concordance with DNA flow cytometry (both P<0.001). We conclude that both section and suspension ISH were able to accurately detect aneuploidy and numerical chromosomal aberrations occurring in larger histological areas. However, section ISH was also capable of revealing (small) focal cytogenetic abnormalities, due to a precise analysis of only target cells. Focal abnormalities were not detected by suspension ISH, probably due to an admixture of non-aberrant tumor cells and stromal elements.

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“…After hybridization, the slides were washed in 50% formamide in 2X SSC (pH 7.0) at room temperature and then washed in 0.1X SSC at 37 ° C. The hybridization signal was detected by the immunoperoxidase technique using the Vectastain ABC kit (Vector) and diaminobenzidine (DAB) as the chromogen substrate, as previously described [12]. Signals were quantitated as previously described [19]. The number of signal spots on a minimum of 100 nuclei in a given area was counted using previously described criteria [19].…”

Section: Chromosomal In Situ Hybridizationmentioning

confidence: 99%

“…Signals were quantitated as previously described [19]. The number of signal spots on a minimum of 100 nuclei in a given area was counted using previously described criteria [19]. A minimum of five randomly chosen areas were counted on each slide from each cell block.…”

Section: Chromosomal In Situ Hybridizationmentioning

confidence: 99%

Technical approach for the study of the genetic evolution of breast cancer from paraffin-embedded tissue sections

Chen

Dhingra

Şahin

et al. 1996

Breast Cancer Res Tr

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We have optimized a technique that allows the study of numerous chromosomal loci (n = 20-50) from single paraffin-embedded tissue sections by microsatellite length polymorphism analysis. DNA samples from normal and breast cancerous tissue can be obtained from the same section by means of microdissection. This technique was further improved by subjecting DNA to several cycles of amplification with a degenerate (universal) primer and then with specific microsatellite primers. This amplified DNA was also used to screen for mutations in the p53 gene by means of PCR-SSCP. In addition adjacent tissue sections were used to assess specific chromosome copy number by interphase cytogenetic analyses (chromosome in situ hybridization) and to analyze expression of specific genes such as p53 and ERBB2. As an example of the use of our approach we performed a detailed chromosome 17 allelotypic analysis in 22 breast tumors (5 ductal carcinomas in situ, 13 invasive ductal carcinomas, and 4 invasive lobular carcinomas). We detected mutations in the p53 gene by PCR-SSCP in 36% of the samples. Samples with significant levels of p53 protein accumulation detected by immunohistochemistry were also positive for mobility shifts in the SSCP analysis. We observed that chromosome 17 allelic losses and imbalance occurred at as early a stage as ductal carcinoma in situ (DCIS). Although in some cases we observed allelic losses or imbalance affecting the 17p13 region, close to the p53 locus, several of the tumors showed dissociation between such loss or imbalance and p53 mutation. Lobular carcinomas were predominantly disomic for chromosome 17 in contrast with ductal tumors, which often showed polysomy for chromosome 17. This comprehensive approach correlating the tumor subtype, its allelotype, with specific chromosome copy number and specific gene mutations and expression in preinvasive or early invasive breast cancer lesions will potentially provide information of relevance for a better understanding of the multistep mechanisms of breast carcinogenesis.

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Quantitative analysis of chromosome in situ hybridization signal in paraffin‐embedded tissue sections

Cited by 36 publications

References 19 publications

3D-confocal structural analysis of bone marrow-derived renal tubular cells during renal ischemia/reperfusion injury

3D-confocal structural analysis of bone marrow-derived renal tubular cells during renal ischemia/reperfusion injury

Interphase in situ hybridization to disaggregated and intact tissue specimens of prostatic adenocarcinomas

Technical approach for the study of the genetic evolution of breast cancer from paraffin-embedded tissue sections

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