Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Ghatak, Atreyi, Combs, Colin K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4251657/
https://www.ncbi.nlm.nih.gov/pubmed/25485234
http://dx.doi.org/10.1016/j.mex.2014.10.007
_version_ 1782347077917868032
author Ghatak, Atreyi
Combs, Colin K.
author_facet Ghatak, Atreyi
Combs, Colin K.
author_sort Ghatak, Atreyi
collection PubMed
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.
format Online
Article
Text
id pubmed-4251657
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher Elsevier
record_format MEDLINE/PubMed
spelling pubmed-42516572015-01-01 Iba1 immunoreactivity is enhanced following an antigen retrieval treatment with EDTA, pH 6.0 Ghatak, Atreyi Combs, Colin K. MethodsX Article 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. Elsevier 2014-11-03 /pmc/articles/PMC4251657/ /pubmed/25485234 http://dx.doi.org/10.1016/j.mex.2014.10.007 Text en © 2014 The Authors http://creativecommons.org/licenses/by/3.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).
spellingShingle Article
Ghatak, Atreyi
Combs, Colin K.
Iba1 immunoreactivity is enhanced following an antigen retrieval treatment with EDTA, pH 6.0
title 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_short 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
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4251657/
https://www.ncbi.nlm.nih.gov/pubmed/25485234
http://dx.doi.org/10.1016/j.mex.2014.10.007
work_keys_str_mv AT ghatakatreyi iba1immunoreactivityisenhancedfollowinganantigenretrievaltreatmentwithedtaph60
AT combscolink iba1immunoreactivityisenhancedfollowinganantigenretrievaltreatmentwithedtaph60