Document Type

Article

Publication Date

11-3-2014

Publication Title

MethodsX

Volume

1

Abstract

Antigen retrieval is a standard procedure to enhance immunohistochemical detection. However, among the many choices of techniquesavailable for antigen retrieval, it is important to choose a method that works specifically for the antibody of interest. The small calciumbinding protein, Iba1, has been well characterized as a microglia specific marker useful for identifying both resting and activatedpopulations (Ito et al., 1998[1]). In this study, we tested whether antigen retrieval methods would increase the sensitivity or improve themorphologic visualization of Iba1 immunoreactive microglia in the brains of wild type C57BL/6 mice and an APP/PS1 mouse model ofAlzheimer’s disease (AD). A more sensitive detection method might allow for better quantitation of microglial changes during disease. Wemodified a protocol which used three different methods and their combination for retrieving specifically anti-Abimmunoreactivity in ADmouse brains to determine whether it improved Iba1 staining (Kai et al., 2012[2]; Murayama et al., 1999[3]). The following modificationswere 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 bathinstead 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 foranti-Abstaining comparison.Iba1 immunoreactivity was best enhanced by boiling in the low pH EDTA solution for both free floating and slide mounted tissues.

First Page

269

Last Page

274

DOI

10.1016/j.mex.2014.10.007

ISSN

22150161

Creative Commons License

Creative Commons Attribution 3.0 License
This work is licensed under a Creative Commons Attribution 3.0 License.

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