Enhanced antigen retrieval of amyloid β immunohistochemistry: re-evaluation of amyloid β pathology in Alzheimer disease and its mouse model

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.

Figure 1.

Enhancement of formic acid (FA)–mediated amyloid β peptide (Aβ) antigen retrieval. Serial brain tissue sections from a 74-year-old male patient with Alzheimer disease (A–D) and from a 13-month-old amyloid precursor protein–Swedish/London (APP-SL) mouse of line 7–5 (E–H) were immunostained with anti-Aβ antibody 4702 following pretreatment by FA alone (FA) (A, E); combination of digestion with proteinase K and FA (PK/FA) (B, F); combination of autoclave heating in EDTA buffer and FA (AC/FA) (C, G); and triple combination of digestion with proteinase K, autoclave heating in EDTA buffer, and FA (PK/AC/FA) (D, H). Pictures (A–D) are from the temporal cortex. Scale bar = 200 µm (A–H).

Figure 2.

General application of the proteinase K digestion (P), EDTA autoclaving (A), and formic acid (FA) treatment (F) (in that order; referred to as “PAF”) method in amyloid β peptide (Aβ) immunohistochemistry. Serial (A–B, C–D, E–F, G–H) brain tissue sections from a 74-year-old male patient with Alzheimer disease (the same patient as shown in Fig. 1) (A–D) and a 16-month-old (E, F) and a 15-month-old (G, H) amyloid precursor protein–Swedish/London (APP-SL) mouse of line 7–5 were immunostained with monoclonal anti-Aβ antibodies 6E10 (A, B, E, F) and 4G8 (C, D, G, H) following pretreatment by the FA (A, C, E, G) and PAF methods (B, D, F, H). Pictures are from the cingulate cortex (A, B) and the frontal cortex (C, D). Scale bar = 200 µm (A–H).

Figure 3.

Amyloid β peptide (Aβ) pathology of Alzheimer disease (AD) brains enhanced by the proteinase K digestion (P), EDTA autoclaving (A), and formic acid (FA) treatment (F) (in that order; referred to as “PAF”) method. Serial brain tissue sections from a 72-year-old female AD patient (the identical patient as shown in Suppl. Fig. S1A,B) (A–D), a 74-year-old male AD patient (the same patient as shown in Fig. 1) (E–H), and a 74-year-old female AD patient (I–L) were immunostained with the 4702 antibody following pretreatment by the FA (A, B, E, F, I, J) and PAF methods (C, D, G, H, K, L). Pictures are from the frontal cortex (A–D), the entorhinal cortex (E–H), and the frontal white matter (I–L). B, D, F, H, J, and L are higher magnification pictures of areas outlined by squares in A, C, E, G, I, and K, respectively. Scale bars = 200µm (A, C, I, K); 50 µm (B, D, F, H); 100µm (E, G); and 20 µm (J, L).

Figure 4.

Effect of amyloid β peptide (Aβ) antigen retrieval (AR) by the proteinase K digestion (P), EDTA autoclaving (A), and formic acid (FA) treatment (F) (in that order; referred to as “PAF”) method over that by the FA method. (A, B) In immunohistochemistry (IHC) with the 4702 antibody, Aβ loads (%) measured in the fusiform cortex of each case from the aged human brains (A) or in the hippocampus of each from the amyloid precursor protein–Swedish/London (APP-SL) mice (B) following the PAF method were plotted against those following the FA method. Significant correlations between the two AR methods were verified both in the aged human brains (Spearman rank correlation coefficient, r _s=0.92; p=9 × 10⁻⁶⁹) and in the mouse brains (r _s=0.80; p=0.003). (C, D) The effect of Aβ AR by the PAF method was significantly higher than that by the FA method both in the aged human and in the mouse brains. In IHC with the 4702 antibody, Aβ-loaded areas measured following the PAF method compared with those following the FA method (set at 1.00; the dotted lines) were 1.59-fold at the 25th percentile, 1.86-fold at the 50th percentile, and 2.31-fold at the 75th percentile in the aged human brains (C) and 3.78-fold at the 25th percentile, 4.64-fold at the 50th percentile, and 12.32-fold at the 75th percentile in the mouse brains (D). (C) Ten (≥3.39) and (D) one (92.79) outliers are not shown. **p=2 × 10⁻²⁸, *p=0.01; analyzed by Wilcoxon signed-rank test. n=162 from 54 individuals where three regions per case examined (A, C), and n=11 (B, D).