A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections

Yun Jiao, Zhiqiang Sun, Teffy Lee, Francesca R. Fusco, Toya D. Kimble, Christopher A. Meade, Sherry Cuthbertson, Anton Reiner

Research output: Contribution to journalArticle

207 Citations (Scopus)

Abstract

The masking of antigens by aldehyde-containing fixatives or by paraffin embedding procedures is a problem for immunohistochemical studies. Enzymatic digestion, formic acid treatment, microwave heating and autoclave heating have been used to deal with this problem, with microwave heating-based antigen retrieval having become widely used as the method of choice. Microwave heating, however, has the shortcoming that it is difficult to precisely control the heating temperature and it is difficult to apply this method of heating to free-floating sections without damaging the sections. We describe here a simple, reliable and sensitive antigen retrieval method that uses water-bath heating. By this method, the temperature can be precisely controlled to yield effective antigen retrieval with minimal tissue damage in free-floating or paraffin-embedded slide-mounted sections. We found that the best results were obtained with a 30 min incubation in a 10-50 mM sodium citrate solution (pH 8.5-9.0) preheated to and maintained at 80°C in a water-bath, followed by 30 min incubation in 0.3-3% nonfat dry milk to reduce nonspecific staining. This method is highly effective for both 40 μm free floating sections, slide-mounted cryostat sections and paraffin-embedded slide-mounted sections, and it works well for tissue from diverse species (human, rat, mouse, pigeon, and zebra finch) and for diverse antigens (e.g. enkephalin, substance P, huntingtin, GluR1, GFAP, and ubiquitin). This method was also found to enhance immunolabeling in glutaraldehyde-fixed tissue that had been prepared for ultrastructural examination, without having a deleterious effect on the ultrastructure.

Original languageEnglish
Pages (from-to)149-162
Number of pages14
JournalJournal of Neuroscience Methods
Volume93
Issue number2
DOIs
Publication statusPublished - Nov 15 1999

Fingerprint

Heating
Antigens
Microwaves
formic acid
Baths
Paraffin
Paraffin Embedding
Finches
Fixatives
Temperature
Water
Equidae
Enkephalins
Columbidae
Glutaral
Substance P
Ubiquitin
Aldehydes
Digestion
Milk

Keywords

  • Antigen retrieval
  • Free-floating sections
  • Immunohistochemistry
  • Paraffin- embedded sections
  • Water-bath heating

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections. / Jiao, Yun; Sun, Zhiqiang; Lee, Teffy; Fusco, Francesca R.; Kimble, Toya D.; Meade, Christopher A.; Cuthbertson, Sherry; Reiner, Anton.

In: Journal of Neuroscience Methods, Vol. 93, No. 2, 15.11.1999, p. 149-162.

Research output: Contribution to journalArticle

Jiao, Yun ; Sun, Zhiqiang ; Lee, Teffy ; Fusco, Francesca R. ; Kimble, Toya D. ; Meade, Christopher A. ; Cuthbertson, Sherry ; Reiner, Anton. / A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections. In: Journal of Neuroscience Methods. 1999 ; Vol. 93, No. 2. pp. 149-162.
@article{84ff288b3d9c43419b3904c35accba04,
title = "A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections",
abstract = "The masking of antigens by aldehyde-containing fixatives or by paraffin embedding procedures is a problem for immunohistochemical studies. Enzymatic digestion, formic acid treatment, microwave heating and autoclave heating have been used to deal with this problem, with microwave heating-based antigen retrieval having become widely used as the method of choice. Microwave heating, however, has the shortcoming that it is difficult to precisely control the heating temperature and it is difficult to apply this method of heating to free-floating sections without damaging the sections. We describe here a simple, reliable and sensitive antigen retrieval method that uses water-bath heating. By this method, the temperature can be precisely controlled to yield effective antigen retrieval with minimal tissue damage in free-floating or paraffin-embedded slide-mounted sections. We found that the best results were obtained with a 30 min incubation in a 10-50 mM sodium citrate solution (pH 8.5-9.0) preheated to and maintained at 80°C in a water-bath, followed by 30 min incubation in 0.3-3{\%} nonfat dry milk to reduce nonspecific staining. This method is highly effective for both 40 μm free floating sections, slide-mounted cryostat sections and paraffin-embedded slide-mounted sections, and it works well for tissue from diverse species (human, rat, mouse, pigeon, and zebra finch) and for diverse antigens (e.g. enkephalin, substance P, huntingtin, GluR1, GFAP, and ubiquitin). This method was also found to enhance immunolabeling in glutaraldehyde-fixed tissue that had been prepared for ultrastructural examination, without having a deleterious effect on the ultrastructure.",
keywords = "Antigen retrieval, Free-floating sections, Immunohistochemistry, Paraffin- embedded sections, Water-bath heating",
author = "Yun Jiao and Zhiqiang Sun and Teffy Lee and Fusco, {Francesca R.} and Kimble, {Toya D.} and Meade, {Christopher A.} and Sherry Cuthbertson and Anton Reiner",
year = "1999",
month = "11",
day = "15",
doi = "10.1016/S0165-0270(99)00142-9",
language = "English",
volume = "93",
pages = "149--162",
journal = "Journal of Neuroscience Methods",
issn = "0165-0270",
publisher = "Elsevier",
number = "2",

}

TY - JOUR

T1 - A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections

AU - Jiao, Yun

AU - Sun, Zhiqiang

AU - Lee, Teffy

AU - Fusco, Francesca R.

AU - Kimble, Toya D.

AU - Meade, Christopher A.

AU - Cuthbertson, Sherry

AU - Reiner, Anton

PY - 1999/11/15

Y1 - 1999/11/15

N2 - The masking of antigens by aldehyde-containing fixatives or by paraffin embedding procedures is a problem for immunohistochemical studies. Enzymatic digestion, formic acid treatment, microwave heating and autoclave heating have been used to deal with this problem, with microwave heating-based antigen retrieval having become widely used as the method of choice. Microwave heating, however, has the shortcoming that it is difficult to precisely control the heating temperature and it is difficult to apply this method of heating to free-floating sections without damaging the sections. We describe here a simple, reliable and sensitive antigen retrieval method that uses water-bath heating. By this method, the temperature can be precisely controlled to yield effective antigen retrieval with minimal tissue damage in free-floating or paraffin-embedded slide-mounted sections. We found that the best results were obtained with a 30 min incubation in a 10-50 mM sodium citrate solution (pH 8.5-9.0) preheated to and maintained at 80°C in a water-bath, followed by 30 min incubation in 0.3-3% nonfat dry milk to reduce nonspecific staining. This method is highly effective for both 40 μm free floating sections, slide-mounted cryostat sections and paraffin-embedded slide-mounted sections, and it works well for tissue from diverse species (human, rat, mouse, pigeon, and zebra finch) and for diverse antigens (e.g. enkephalin, substance P, huntingtin, GluR1, GFAP, and ubiquitin). This method was also found to enhance immunolabeling in glutaraldehyde-fixed tissue that had been prepared for ultrastructural examination, without having a deleterious effect on the ultrastructure.

AB - The masking of antigens by aldehyde-containing fixatives or by paraffin embedding procedures is a problem for immunohistochemical studies. Enzymatic digestion, formic acid treatment, microwave heating and autoclave heating have been used to deal with this problem, with microwave heating-based antigen retrieval having become widely used as the method of choice. Microwave heating, however, has the shortcoming that it is difficult to precisely control the heating temperature and it is difficult to apply this method of heating to free-floating sections without damaging the sections. We describe here a simple, reliable and sensitive antigen retrieval method that uses water-bath heating. By this method, the temperature can be precisely controlled to yield effective antigen retrieval with minimal tissue damage in free-floating or paraffin-embedded slide-mounted sections. We found that the best results were obtained with a 30 min incubation in a 10-50 mM sodium citrate solution (pH 8.5-9.0) preheated to and maintained at 80°C in a water-bath, followed by 30 min incubation in 0.3-3% nonfat dry milk to reduce nonspecific staining. This method is highly effective for both 40 μm free floating sections, slide-mounted cryostat sections and paraffin-embedded slide-mounted sections, and it works well for tissue from diverse species (human, rat, mouse, pigeon, and zebra finch) and for diverse antigens (e.g. enkephalin, substance P, huntingtin, GluR1, GFAP, and ubiquitin). This method was also found to enhance immunolabeling in glutaraldehyde-fixed tissue that had been prepared for ultrastructural examination, without having a deleterious effect on the ultrastructure.

KW - Antigen retrieval

KW - Free-floating sections

KW - Immunohistochemistry

KW - Paraffin- embedded sections

KW - Water-bath heating

UR - http://www.scopus.com/inward/record.url?scp=0032776743&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032776743&partnerID=8YFLogxK

U2 - 10.1016/S0165-0270(99)00142-9

DO - 10.1016/S0165-0270(99)00142-9

M3 - Article

C2 - 10634500

AN - SCOPUS:0032776743

VL - 93

SP - 149

EP - 162

JO - Journal of Neuroscience Methods

JF - Journal of Neuroscience Methods

SN - 0165-0270

IS - 2

ER -