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). 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; Murayama et al., 1999). 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.
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Atreyi Ghatak and Colin K. Combs. "Iba1 immunoreactivity is enhanced following an antigen retrieval treatment with EDTA, pH 6.0" (2014). Biomedical Sciences Faculty Publications. 19.