Progress in focus: recent advances in Histochemistry and Cell Biology

Asan, Esther

doi:10.1007/s00418-002-0480-5

Cited by 4 publications

(1 citation statement)

References 173 publications

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“…However, the detection sensitivity of ISH is sometimes limited. The combination of these two methods, called in situ PCR, first described by Haase et al in 1990 [ 6 ], is an extremely highly sensitive technique to localize a single gene copy at the individual cell level [ 1 – 4 , 11 – 13 , 20 , 22 ]. However, in situ PCR has not gained wide acceptance by the scientific community, partly because of the significant variations in the optimal conditions of the protocol, which depend on the tissues used for analysis [ 11 , 12 ].…”

Section: Introductionmentioning

confidence: 99%

Improvement of In Situ PCR by Optimization of PCR Cycle Number and Proteinase K Concentration: Localization of X Chromosome-Linked Phosphoglycerate Kinase-1 Gene in Mouse Reproductive Organs

Hishikawa

Yamamoto‐Fukuda

et al. 2009

Acta Histochem. Cytochem

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In situ polymerase chain reaction (in situ PCR), which can detect a few copies of genes within a cell by amplifying the target gene, was developed to better understand the biological functions of tissues. In this study, we optimized the protocol conditions for the detection of X chromosome-linked phosphoglycerate kinase-1 (pgk-1) gene in paraffin-embedded sections of mouse reproductive organs. The effects of various concentrations of proteinase K (PK) and PCR cycle numbers were examined. To label the amplified DNA, we used digoxigenindUTP (Dig), Cy-3-dUTP (Cy-3), or FluorX-dCTP (FluorX). The optimal concentration of PK was 50 µg/ml for the ovary and 10 µg/ml for the testis. Ten PCR cycles were optimal for Dig and 25 cycles were optimal for FluorX and Cy-3 in the ovary and testis. The signal-to-noise ratio of FluorX and Cy-3 for ovarian tissue was better than that of Dig. Using the above conditions, we detected 1-4 and 1-2 spots of pgk-1 in the nuclei of granulosa and germ cells, respectively. Our results indicate that in situ PCR is useful for detecting a specific gene in paraffin-embedded sections under optimized conditions of both PCR cycle number and PK concentration.

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Section: Introductionmentioning

confidence: 99%

Improvement of In Situ PCR by Optimization of PCR Cycle Number and Proteinase K Concentration: Localization of X Chromosome-Linked Phosphoglycerate Kinase-1 Gene in Mouse Reproductive Organs

Hishikawa

Yamamoto‐Fukuda

et al. 2009

Acta Histochem. Cytochem

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Innovative techniques and applications in histochemistry and cell biology

Asan

2003

Histochemistry and Cell Biology

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Recent studies documenting novel histochemical methods and applications in cell biology and in other areas of the life sciences have again rendered insights into structure and functions of tissues, cells, and cellular components to the level of proteins and genes. Particularly, sophisticated microscopic techniques have proved to be able to significantly advance our knowledge. Findings of recent investigations representing this progress are summarized in the present review.

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News and views in Histochemistry and Cell Biology

Asan¹,

Drenckhahn²

2004

Histochem Cell Biol

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Advances in histochemical methodology and ingenious applications of novel and improved methods continue to confirm the standing of morphological means and approaches in research efforts, and contribute significantly to increasing our knowledge about structures and functions in all areas of the life sciences from cell biology to pathology. Reports published during recent months documenting this progress are summarized in the present review.

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Progress in focus: recent advances in Histochemistry and Cell Biology

Cited by 4 publications

References 173 publications

Improvement of In Situ PCR by Optimization of PCR Cycle Number and Proteinase K Concentration: Localization of X Chromosome-Linked Phosphoglycerate Kinase-1 Gene in Mouse Reproductive Organs

Improvement of In Situ PCR by Optimization of PCR Cycle Number and Proteinase K Concentration: Localization of X Chromosome-Linked Phosphoglycerate Kinase-1 Gene in Mouse Reproductive Organs

Innovative techniques and applications in histochemistry and cell biology

News and views in Histochemistry and Cell Biology

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