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 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.

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