Application of heat-induced antigen retrieval to aldehyde-fixed fresh frozen sections

Shuji Yamashita, Yasunori Okada

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

We applied the heat-induced antigen retrieval (HIAR) to aldehyde-fixed fresh frozen sections based on a new approach (i.e., a rapid and complete immobilization of antigen followed by heating). Frozen sections were fixed with 10% formalin in 0.1 M cacodylate buffer (pH 7.4) containing 25 mM CaCl 2 for 30 min, or with 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 min at room temperature, and then autoclaved in 20 mM Tris-HCl buffer (pH 9.0) for 10 min at 120C. Both fixatives yielded good tissue structure after autoclaving. In the sections fixed with formalin containing CaCl 2, 20 of 22 antigens located in the nucleus, cytoplasm, membranes, and extracellular matrix greatly recovered their antigenicity after autoclaving; only two antigens exhibited stronger immunoreaction in acetone-fixed fresh frozen sections than these sections. Heating also retrieved the immunoreactivity of at least 14 antigens in the sections fixed with glutaraldehyde. We used the similar procedures to localize ligand-free estrogen receptor α (ERα) and glucocorticoid receptors (GR). Mouse uterine cells exhibited almost the same nuclear ERα immunostaining regardless of the hormonal status in glutaraldehyde-fixed fresh frozen sections and unliganded GR was localized mainly in the nucleus of mouse hepatocytes in fresh frozen sections fixed with 20% formalin containing 50 or 75 mM CaCl2 at 40C, after autoclaving. These results demonstrate that HIAR is useful for the immunohistochemistry of many antigens in aldehyde-fixed fresh frozen sections.

Original languageEnglish
Pages (from-to)1421-1432
Number of pages12
JournalJournal of Histochemistry and Cytochemistry
Volume53
Issue number11
DOIs
Publication statusPublished - 2005 Nov

Fingerprint

Frozen Sections
Aldehydes
Hot Temperature
Antigens
Glutaral
Formaldehyde
Glucocorticoid Receptors
Estrogen Receptors
Heating
Buffers
Cacodylic Acid
Fixatives
Tromethamine
Acetone
Extracellular Matrix
Hepatocytes
Cytoplasm
Immunohistochemistry
Phosphates
Ligands

Keywords

  • Fresh frozen section
  • Heat-induced antigen retrieval
  • Immunohistochemistry
  • Unoccupied steroid hormone receptors

ASJC Scopus subject areas

  • Anatomy
  • Cell Biology

Cite this

Application of heat-induced antigen retrieval to aldehyde-fixed fresh frozen sections. / Yamashita, Shuji; Okada, Yasunori.

In: Journal of Histochemistry and Cytochemistry, Vol. 53, No. 11, 11.2005, p. 1421-1432.

Research output: Contribution to journalArticle

@article{e291896c26aa4ed68bf5221544b03b12,
title = "Application of heat-induced antigen retrieval to aldehyde-fixed fresh frozen sections",
abstract = "We applied the heat-induced antigen retrieval (HIAR) to aldehyde-fixed fresh frozen sections based on a new approach (i.e., a rapid and complete immobilization of antigen followed by heating). Frozen sections were fixed with 10{\%} formalin in 0.1 M cacodylate buffer (pH 7.4) containing 25 mM CaCl 2 for 30 min, or with 0.5{\%} glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 min at room temperature, and then autoclaved in 20 mM Tris-HCl buffer (pH 9.0) for 10 min at 120C. Both fixatives yielded good tissue structure after autoclaving. In the sections fixed with formalin containing CaCl 2, 20 of 22 antigens located in the nucleus, cytoplasm, membranes, and extracellular matrix greatly recovered their antigenicity after autoclaving; only two antigens exhibited stronger immunoreaction in acetone-fixed fresh frozen sections than these sections. Heating also retrieved the immunoreactivity of at least 14 antigens in the sections fixed with glutaraldehyde. We used the similar procedures to localize ligand-free estrogen receptor α (ERα) and glucocorticoid receptors (GR). Mouse uterine cells exhibited almost the same nuclear ERα immunostaining regardless of the hormonal status in glutaraldehyde-fixed fresh frozen sections and unliganded GR was localized mainly in the nucleus of mouse hepatocytes in fresh frozen sections fixed with 20{\%} formalin containing 50 or 75 mM CaCl2 at 40C, after autoclaving. These results demonstrate that HIAR is useful for the immunohistochemistry of many antigens in aldehyde-fixed fresh frozen sections.",
keywords = "Fresh frozen section, Heat-induced antigen retrieval, Immunohistochemistry, Unoccupied steroid hormone receptors",
author = "Shuji Yamashita and Yasunori Okada",
year = "2005",
month = "11",
doi = "10.1369/jhc.4A6579.2005",
language = "English",
volume = "53",
pages = "1421--1432",
journal = "Journal of Histochemistry and Cytochemistry",
issn = "0022-1554",
publisher = "Histochemical Society Inc.",
number = "11",

}

TY - JOUR

T1 - Application of heat-induced antigen retrieval to aldehyde-fixed fresh frozen sections

AU - Yamashita, Shuji

AU - Okada, Yasunori

PY - 2005/11

Y1 - 2005/11

N2 - We applied the heat-induced antigen retrieval (HIAR) to aldehyde-fixed fresh frozen sections based on a new approach (i.e., a rapid and complete immobilization of antigen followed by heating). Frozen sections were fixed with 10% formalin in 0.1 M cacodylate buffer (pH 7.4) containing 25 mM CaCl 2 for 30 min, or with 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 min at room temperature, and then autoclaved in 20 mM Tris-HCl buffer (pH 9.0) for 10 min at 120C. Both fixatives yielded good tissue structure after autoclaving. In the sections fixed with formalin containing CaCl 2, 20 of 22 antigens located in the nucleus, cytoplasm, membranes, and extracellular matrix greatly recovered their antigenicity after autoclaving; only two antigens exhibited stronger immunoreaction in acetone-fixed fresh frozen sections than these sections. Heating also retrieved the immunoreactivity of at least 14 antigens in the sections fixed with glutaraldehyde. We used the similar procedures to localize ligand-free estrogen receptor α (ERα) and glucocorticoid receptors (GR). Mouse uterine cells exhibited almost the same nuclear ERα immunostaining regardless of the hormonal status in glutaraldehyde-fixed fresh frozen sections and unliganded GR was localized mainly in the nucleus of mouse hepatocytes in fresh frozen sections fixed with 20% formalin containing 50 or 75 mM CaCl2 at 40C, after autoclaving. These results demonstrate that HIAR is useful for the immunohistochemistry of many antigens in aldehyde-fixed fresh frozen sections.

AB - We applied the heat-induced antigen retrieval (HIAR) to aldehyde-fixed fresh frozen sections based on a new approach (i.e., a rapid and complete immobilization of antigen followed by heating). Frozen sections were fixed with 10% formalin in 0.1 M cacodylate buffer (pH 7.4) containing 25 mM CaCl 2 for 30 min, or with 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 min at room temperature, and then autoclaved in 20 mM Tris-HCl buffer (pH 9.0) for 10 min at 120C. Both fixatives yielded good tissue structure after autoclaving. In the sections fixed with formalin containing CaCl 2, 20 of 22 antigens located in the nucleus, cytoplasm, membranes, and extracellular matrix greatly recovered their antigenicity after autoclaving; only two antigens exhibited stronger immunoreaction in acetone-fixed fresh frozen sections than these sections. Heating also retrieved the immunoreactivity of at least 14 antigens in the sections fixed with glutaraldehyde. We used the similar procedures to localize ligand-free estrogen receptor α (ERα) and glucocorticoid receptors (GR). Mouse uterine cells exhibited almost the same nuclear ERα immunostaining regardless of the hormonal status in glutaraldehyde-fixed fresh frozen sections and unliganded GR was localized mainly in the nucleus of mouse hepatocytes in fresh frozen sections fixed with 20% formalin containing 50 or 75 mM CaCl2 at 40C, after autoclaving. These results demonstrate that HIAR is useful for the immunohistochemistry of many antigens in aldehyde-fixed fresh frozen sections.

KW - Fresh frozen section

KW - Heat-induced antigen retrieval

KW - Immunohistochemistry

KW - Unoccupied steroid hormone receptors

UR - http://www.scopus.com/inward/record.url?scp=27244433299&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=27244433299&partnerID=8YFLogxK

U2 - 10.1369/jhc.4A6579.2005

DO - 10.1369/jhc.4A6579.2005

M3 - Article

C2 - 16046672

AN - SCOPUS:27244433299

VL - 53

SP - 1421

EP - 1432

JO - Journal of Histochemistry and Cytochemistry

JF - Journal of Histochemistry and Cytochemistry

SN - 0022-1554

IS - 11

ER -