Heat-induced Antigen Retrieval in Conventionally Processed Epon-embedded Specimens: Procedures and Mechanisms

Shuji Yamashita, Yasunori Okada

研究成果: Article

11 引用 (Scopus)

抄録

We studied the effectiveness of heat-induced antigen retrieval (HIAR) in conventionally processed, epon-embedded specimens and the mechanisms of HIAR in the specimens. Frozen sections were first immunostained to examine the possibility of using HIAR for 18 antigens to avoid the effects of epoxy resin embedment. The antigenicity of 7 out of 18 antigens was retrieved with glutaraldehyde fixation followed by osmium tetroxide treatment whereas none were retrieved with glutaraldehyde fixation without post-osmication. Six antigens also exhibited positive immunostaining in semi-thin epon sections when the sections were deplasticized with sodium ethoxide followed by autoclaving. In the immunoelectron microscopy with the post-embedding method, positive reactions with fine ultrastructures were obtained using HIAR without deplasticization. These results suggested that osmium tetroxide binds to ethylene double bonds (which are introduced into protein crosslinks by glutaraldehyde) and forms an extremely stable resonance interaction with the Schiff bases, thus destabilizing the protein crosslinks. Heating also further degrades these crosslinks. The present study demonstrated that archival epon blocks can be useful resources for immunohistochemical studies for both light and electron microscopy.

元の言語English
ページ(範囲)584-597
ページ数14
ジャーナルJournal of Histochemistry and Cytochemistry
62
発行部数8
DOI
出版物ステータスPublished - 2014

Fingerprint

Hot Temperature
Antigens
Glutaral
Osmium Tetroxide
Epoxy Resins
Immunoelectron Microscopy
Schiff Bases
Frozen Sections
EPON
Heating
Electron Microscopy
Proteins
Light

ASJC Scopus subject areas

  • Anatomy
  • Histology

これを引用

@article{dd3d8a7a15124f94b38cc535ed20ac6c,
title = "Heat-induced Antigen Retrieval in Conventionally Processed Epon-embedded Specimens: Procedures and Mechanisms",
abstract = "We studied the effectiveness of heat-induced antigen retrieval (HIAR) in conventionally processed, epon-embedded specimens and the mechanisms of HIAR in the specimens. Frozen sections were first immunostained to examine the possibility of using HIAR for 18 antigens to avoid the effects of epoxy resin embedment. The antigenicity of 7 out of 18 antigens was retrieved with glutaraldehyde fixation followed by osmium tetroxide treatment whereas none were retrieved with glutaraldehyde fixation without post-osmication. Six antigens also exhibited positive immunostaining in semi-thin epon sections when the sections were deplasticized with sodium ethoxide followed by autoclaving. In the immunoelectron microscopy with the post-embedding method, positive reactions with fine ultrastructures were obtained using HIAR without deplasticization. These results suggested that osmium tetroxide binds to ethylene double bonds (which are introduced into protein crosslinks by glutaraldehyde) and forms an extremely stable resonance interaction with the Schiff bases, thus destabilizing the protein crosslinks. Heating also further degrades these crosslinks. The present study demonstrated that archival epon blocks can be useful resources for immunohistochemical studies for both light and electron microscopy.",
keywords = "heat-induced antigen retrieval, immunoelectron microscopy, mechanism of antigen retrieval, osmicated and epon-embedded specimen",
author = "Shuji Yamashita and Yasunori Okada",
year = "2014",
doi = "10.1369/0022155414537899",
language = "English",
volume = "62",
pages = "584--597",
journal = "Journal of Histochemistry and Cytochemistry",
issn = "0022-1554",
publisher = "Histochemical Society Inc.",
number = "8",

}

TY - JOUR

T1 - Heat-induced Antigen Retrieval in Conventionally Processed Epon-embedded Specimens

T2 - Procedures and Mechanisms

AU - Yamashita, Shuji

AU - Okada, Yasunori

PY - 2014

Y1 - 2014

N2 - We studied the effectiveness of heat-induced antigen retrieval (HIAR) in conventionally processed, epon-embedded specimens and the mechanisms of HIAR in the specimens. Frozen sections were first immunostained to examine the possibility of using HIAR for 18 antigens to avoid the effects of epoxy resin embedment. The antigenicity of 7 out of 18 antigens was retrieved with glutaraldehyde fixation followed by osmium tetroxide treatment whereas none were retrieved with glutaraldehyde fixation without post-osmication. Six antigens also exhibited positive immunostaining in semi-thin epon sections when the sections were deplasticized with sodium ethoxide followed by autoclaving. In the immunoelectron microscopy with the post-embedding method, positive reactions with fine ultrastructures were obtained using HIAR without deplasticization. These results suggested that osmium tetroxide binds to ethylene double bonds (which are introduced into protein crosslinks by glutaraldehyde) and forms an extremely stable resonance interaction with the Schiff bases, thus destabilizing the protein crosslinks. Heating also further degrades these crosslinks. The present study demonstrated that archival epon blocks can be useful resources for immunohistochemical studies for both light and electron microscopy.

AB - We studied the effectiveness of heat-induced antigen retrieval (HIAR) in conventionally processed, epon-embedded specimens and the mechanisms of HIAR in the specimens. Frozen sections were first immunostained to examine the possibility of using HIAR for 18 antigens to avoid the effects of epoxy resin embedment. The antigenicity of 7 out of 18 antigens was retrieved with glutaraldehyde fixation followed by osmium tetroxide treatment whereas none were retrieved with glutaraldehyde fixation without post-osmication. Six antigens also exhibited positive immunostaining in semi-thin epon sections when the sections were deplasticized with sodium ethoxide followed by autoclaving. In the immunoelectron microscopy with the post-embedding method, positive reactions with fine ultrastructures were obtained using HIAR without deplasticization. These results suggested that osmium tetroxide binds to ethylene double bonds (which are introduced into protein crosslinks by glutaraldehyde) and forms an extremely stable resonance interaction with the Schiff bases, thus destabilizing the protein crosslinks. Heating also further degrades these crosslinks. The present study demonstrated that archival epon blocks can be useful resources for immunohistochemical studies for both light and electron microscopy.

KW - heat-induced antigen retrieval

KW - immunoelectron microscopy

KW - mechanism of antigen retrieval

KW - osmicated and epon-embedded specimen

UR - http://www.scopus.com/inward/record.url?scp=84905047374&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84905047374&partnerID=8YFLogxK

U2 - 10.1369/0022155414537899

DO - 10.1369/0022155414537899

M3 - Article

AN - SCOPUS:84905047374

VL - 62

SP - 584

EP - 597

JO - Journal of Histochemistry and Cytochemistry

JF - Journal of Histochemistry and Cytochemistry

SN - 0022-1554

IS - 8

ER -