Iba1 immunoreactivity is enhanced following an antigen retrieval treatment with EDTA, pH 6.0
Antigen retrieval is a standard procedure to enhance immunohistochemical detection. However, among the many choices of techniques available for antigen retrieval, it is important to choose a method that works specifically for the antibody of interest. The small calcium binding protein, Iba1, has bee...
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doaj-341030cdbc96443890db67ecaea333e02020-11-25T02:02:58ZengElsevierMethodsX2215-01612014-01-011C26927410.1016/j.mex.2014.10.007Iba1 immunoreactivity is enhanced following an antigen retrieval treatment with EDTA, pH 6.0Atreyi GhatakColin K. CombsAntigen retrieval is a standard procedure to enhance immunohistochemical detection. However, among the many choices of techniques available for antigen retrieval, it is important to choose a method that works specifically for the antibody of interest. The small calcium binding protein, Iba1, has been well characterized as a microglia specific marker useful for identifying both resting and activated populations (Ito et al., 1998 [1]). In this study, we tested whether antigen retrieval methods would increase the sensitivity or improve the morphologic visualization of Iba1 immunoreactive microglia in the brains of wild type C57BL/6 mice and an APP/PS1 mouse model of Alzheimer's disease (AD). A more sensitive detection method might allow for better quantitation of microglial changes during disease. We modified a protocol which used three different methods and their combination for retrieving specifically anti-Aβ immunoreactivity in AD mouse brains to determine whether it improved Iba1 staining (Kai et al., 2012 [2]; Murayama et al., 1999 [3]). The following modifications were made to the original protocol: 1. We boiled the free floating brain sections or slide mounted brain sections in 10 mM EDTA solution (pH 6.0) in a secondary water bath instead of autoclaving for attempting Iba1 antigen retrieval. 2. We used a 15 min, 0.25% trypsin-EDTA treatment instead of protease K for attempting Iba1 antigen retrieval. 3. We immunostained with anti-Iba1 antibody as our primary interest, but also stained some sections in parallel with 4G8 antibody for anti-Aβ staining comparison. Iba1 immunoreactivity was best enhanced by boiling in the low pH EDTA solution for both free floating and slide mounted tissues.http://www.sciencedirect.com/science/article/pii/S2215016114000211Antigen retrieval |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Atreyi Ghatak Colin K. Combs |
spellingShingle |
Atreyi Ghatak Colin K. Combs Iba1 immunoreactivity is enhanced following an antigen retrieval treatment with EDTA, pH 6.0 MethodsX Antigen retrieval |
author_facet |
Atreyi Ghatak Colin K. Combs |
author_sort |
Atreyi Ghatak |
title |
Iba1 immunoreactivity is enhanced following an antigen retrieval treatment with EDTA, pH 6.0 |
title_short |
Iba1 immunoreactivity is enhanced following an antigen retrieval treatment with EDTA, pH 6.0 |
title_full |
Iba1 immunoreactivity is enhanced following an antigen retrieval treatment with EDTA, pH 6.0 |
title_fullStr |
Iba1 immunoreactivity is enhanced following an antigen retrieval treatment with EDTA, pH 6.0 |
title_full_unstemmed |
Iba1 immunoreactivity is enhanced following an antigen retrieval treatment with EDTA, pH 6.0 |
title_sort |
iba1 immunoreactivity is enhanced following an antigen retrieval treatment with edta, ph 6.0 |
publisher |
Elsevier |
series |
MethodsX |
issn |
2215-0161 |
publishDate |
2014-01-01 |
description |
Antigen retrieval is a standard procedure to enhance immunohistochemical detection. However, among the many choices of techniques available for antigen retrieval, it is important to choose a method that works specifically for the antibody of interest. The small calcium binding protein, Iba1, has been well characterized as a microglia specific marker useful for identifying both resting and activated populations (Ito et al., 1998 [1]). In this study, we tested whether antigen retrieval methods would increase the sensitivity or improve the morphologic visualization of Iba1 immunoreactive microglia in the brains of wild type C57BL/6 mice and an APP/PS1 mouse model of Alzheimer's disease (AD). A more sensitive detection method might allow for better quantitation of microglial changes during disease. We modified a protocol which used three different methods and their combination for retrieving specifically anti-Aβ immunoreactivity in AD mouse brains to determine whether it improved Iba1 staining (Kai et al., 2012 [2]; Murayama et al., 1999 [3]). The following modifications were made to the original protocol:
1.
We boiled the free floating brain sections or slide mounted brain sections in 10 mM EDTA solution (pH 6.0) in a secondary water bath instead of autoclaving for attempting Iba1 antigen retrieval.
2.
We used a 15 min, 0.25% trypsin-EDTA treatment instead of protease K for attempting Iba1 antigen retrieval.
3.
We immunostained with anti-Iba1 antibody as our primary interest, but also stained some sections in parallel with 4G8 antibody for anti-Aβ staining comparison.
Iba1 immunoreactivity was best enhanced by boiling in the low pH EDTA solution for both free floating and slide mounted tissues. |
topic |
Antigen retrieval |
url |
http://www.sciencedirect.com/science/article/pii/S2215016114000211 |
work_keys_str_mv |
AT atreyighatak iba1immunoreactivityisenhancedfollowinganantigenretrievaltreatmentwithedtaph60 AT colinkcombs iba1immunoreactivityisenhancedfollowinganantigenretrievaltreatmentwithedtaph60 |
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