Heat-assisted stretching of paraffin sections on hot plate weakens immunoreactivity of orotate phosphoribosyltransferase

Shingo Kamoshida, Nao Sakamoto, Hiroshi Matsuoka, Yoichi Sakurai, Kazuki Sakamoto, Yoshikazu Sugimoto, Masakazu Fukushima, Yutaka Tsutsumi

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Orotate phosphoribosyltransferase (OPRT) is the key enzyme for the phosphorylation of 5-fluorouracil (5-FU), the rate-limiting step for acquiring its anti-tumor effect Since high enzyme activities and rnRNA levels of OPRT are said to be associated with 5-FU chemosensitivity of cancer cells, the immunohistochemical demonstration of OPRT can be expected to contribute to the selection of patients who suffer from 5-FU-sensitive cancer. During a study for establishing the appropriate immunostaining condition using rabbit antiserum in formalin-fixed, paraffin-embedded sections of human cancer, we unexpectedly uncovered the fact that heat-assisted stretching of paraffin sections on a hot plate just after sectioning was critical for preserving OPRT immunoreactivity; namely, stretching sections briefly at 70°C or higher significantly reduced the immunoreactivity. Overnight drying of the sections in an oven at 37°C or 60°C did not influence the immunoreactivity, but pretreatments, including 0.2% trypsin, 0.002% proteinase K, and pressure cooking in 10 mM citrate buffer, pH 6.0 and pH 7.0, and 1 mM ethylenediaminetetraacetic acid solution, pH 8.0, seriously deteriorated the OPRT epitopes. The immunoreactivity was relatively resistant to overfixation, though weakened after fixation in formalin for four weeks. Xenografts with high OPRT enzyme activities showed distinct positive cytoplasmic staining, but those with low OPRT activities were equivocal. OPRT expression in routinely processed cancer tissues also provided valuable data.

Original languageEnglish
Pages (from-to)69-74
Number of pages6
JournalActa Histochemica et Cytochemica
Volume38
Issue number1
DOIs
Publication statusPublished - 2005
Externally publishedYes

Fingerprint

Orotate Phosphoribosyltransferase
Paraffin
Stretching
Hot Temperature
Fluorouracil
Enzyme activity
Neoplasms
Formaldehyde
Enzymes
Endopeptidase K
Phosphorylation
Cooking
Ovens
Heterografts
Edetic Acid
Citric Acid
Trypsin
Patient Selection
Tumors
Immune Sera

Keywords

  • Fixation period
  • Heat-assisted stretching
  • Immunohistochemistry
  • Orotate phosphoribosyltransferase
  • Paraffin section

ASJC Scopus subject areas

  • Biochemistry
  • Neuroscience(all)
  • Endocrinology
  • Anatomy
  • Cell Biology

Cite this

Heat-assisted stretching of paraffin sections on hot plate weakens immunoreactivity of orotate phosphoribosyltransferase. / Kamoshida, Shingo; Sakamoto, Nao; Matsuoka, Hiroshi; Sakurai, Yoichi; Sakamoto, Kazuki; Sugimoto, Yoshikazu; Fukushima, Masakazu; Tsutsumi, Yutaka.

In: Acta Histochemica et Cytochemica, Vol. 38, No. 1, 2005, p. 69-74.

Research output: Contribution to journalArticle

Kamoshida, Shingo ; Sakamoto, Nao ; Matsuoka, Hiroshi ; Sakurai, Yoichi ; Sakamoto, Kazuki ; Sugimoto, Yoshikazu ; Fukushima, Masakazu ; Tsutsumi, Yutaka. / Heat-assisted stretching of paraffin sections on hot plate weakens immunoreactivity of orotate phosphoribosyltransferase. In: Acta Histochemica et Cytochemica. 2005 ; Vol. 38, No. 1. pp. 69-74.
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AU - Sugimoto, Yoshikazu

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AU - Tsutsumi, Yutaka

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AB - Orotate phosphoribosyltransferase (OPRT) is the key enzyme for the phosphorylation of 5-fluorouracil (5-FU), the rate-limiting step for acquiring its anti-tumor effect Since high enzyme activities and rnRNA levels of OPRT are said to be associated with 5-FU chemosensitivity of cancer cells, the immunohistochemical demonstration of OPRT can be expected to contribute to the selection of patients who suffer from 5-FU-sensitive cancer. During a study for establishing the appropriate immunostaining condition using rabbit antiserum in formalin-fixed, paraffin-embedded sections of human cancer, we unexpectedly uncovered the fact that heat-assisted stretching of paraffin sections on a hot plate just after sectioning was critical for preserving OPRT immunoreactivity; namely, stretching sections briefly at 70°C or higher significantly reduced the immunoreactivity. Overnight drying of the sections in an oven at 37°C or 60°C did not influence the immunoreactivity, but pretreatments, including 0.2% trypsin, 0.002% proteinase K, and pressure cooking in 10 mM citrate buffer, pH 6.0 and pH 7.0, and 1 mM ethylenediaminetetraacetic acid solution, pH 8.0, seriously deteriorated the OPRT epitopes. The immunoreactivity was relatively resistant to overfixation, though weakened after fixation in formalin for four weeks. Xenografts with high OPRT enzyme activities showed distinct positive cytoplasmic staining, but those with low OPRT activities were equivocal. OPRT expression in routinely processed cancer tissues also provided valuable data.

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