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 differentmethods and their combination for retrieving specifically anti-Ab 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: We boiled the free floating brain sections or slide mounted brain sections in 10mM EDTA solution (pH 6.0) in a secondary water bath instead of autoclaving for attempting Iba1 antigen retrieval. We used a 15min, 0.25% trypsin-EDTA treatment instead of protease K for attempting Iba1 antigen retrieval. We immunostained with anti-Iba1 antibody as our primary interest, but also stained some sections in parallel with 4G8 antibody for anti-Ab staining comparison. Iba1 immunoreactivity was best enhanced by boiling in the low pH EDTA solution for both free floating and slide mounted tissues. (C) 2014 The Authors. Published by Elsevier B.V.