Enhanced Antigen Retrieval of Amyloid β Immunohistochemistry

Abstract

Senile plaques, extracellular deposits of amyloid β peptide (Aβ), are one of the pathological hallmarks of Alzheimer disease (AD). As the standard immunohistochemical detection method for Aβ deposits, anti-Aβ immunohistochemistry combined with antigen retrieval (AR) by formic acid (FA) has been generally used. Here, we present a more efficient AR for Aβ antigen. On brain sections of AD and its mouse model, a double combination of either autoclave heating in EDTA buffer or digestion with proteinase K plus FA treatment reinforced Aβ immunoreactivity. A further triple combination of digestion with proteinase K (P), autoclave heating in EDTA buffer (A), and FA treatment (F), when employed in this order, gave a more enhanced immunoreactivity. Our PAF method prominently visualized various forms of Aβ deposits in AD that have not been clearly detected previously and revealed numerous minute-sized plaques both in AD and the mouse model. Quantification of Aβ loads showed that the AR effect by the PAF method was 1.86-fold (in the aged human brain) and 4.64-fold (in the mouse brain) higher than that by the FA method. Thus, the PAF method could have the potential to be the most sensitive tool so far to study Aβ pathology in AD and its mouse model.

Lesion Profiling and Subcellular Prion Localization of Cervid Chronic Wasting Disease in Domestic Cats

“…Mechanistically, it has been proposed that the proteolytic digestion of blocking proteins that surround amyloid fibrils might occur and thereby enhance subsequent antigen retrieval and antibody binding steps. 26 Our demonstration of improved IHC PrP D detection using a combination of FA, HIER, and PK is similar to reports using the L42 antibody in scrapie-infected sheep. 20 …”

“…To overcome this problem, we modified our previous protocol, which combined PLP fixation, 88% FA immersion, and HIER, to include proteinase digestion, based on a recent publication describing amyloid β detection in a mouse model. 26 Among the aldehyde fixatives, PLP has previously been shown to enhance the sensitivity of IHC-based prion detection techniques. 36,43 The antigen retrieval benefits of FA are believed to be the result of acidic hydrolysis, which is proposed to result in the unfolding of amyloid polymers.…”

Lack of spontaneous age-related brain pathology in Octodon degus: a reappraisal of the model

“…1B). Finally, we performed a sensitive immunohistochemistry to detect Aβ deposition16 (Fig. 7) and/or hyperphosphorylated tau staining (Supp.…”

“…Grey level quantification or immunostaining-positive surface quantification were performed as previously described23. To assess the presence of amyloid β extracellular deposits, the mouse 6E10 anti β -amyloid clone antibody (Eurogentec, 6E10, 1:1000) was used as previously reported16. Briefly, 50 μm sagittal free-floating sections of representative levels of octodons at different ages and APP/PS1 at age 6–9 months were pretreated with a further triple combination of digestion with 1 μg/mL proteinase K solution (10 min RT), water-bath EDTA heating (20 min −80 °C) and 98% formic acid treatment (1 min RT).…”

Iba1 immunoreactivity is enhanced following an antigen retrieval treatment with EDTA, pH 6.0

“…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: 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.…”