Fig. 5

ApoE, CFH, and HTRA1 protein levels in AAT^–/– mice. (a) Immunofluorescence on retinal cross sections of 2 M old (first and second column) and 18-24 M old mice (third and fourth column) comparing AAT^+/+ (first and third column) to AAT^–/– littermates (second and fourth column). Apolipoprotein E (ApoE; green) displays no signal at the BrM/RPE in 2 M old mice but shows a much stronger signal at the BrM/RPE in aged wildtype mice (white arrows) compared to the aged AAT^–/– mice. To the right is a western blot analysis of ApoE protein levels with RPE/choroid tissue extracts from AAT^+/+ (black bar) and AAT^–/– mice (white bar) at 18-24 M to quantify the changes in protein levels seen by antibody staining on sections. Example of western blot bands are shown beneath the bar graph. (b) Immunofluorescence on retinal cross sections and western blot analysis following the same format as shown in panel (a) for complement factor H (CFH; red) showing an increase in signal at the BrM/RPE (white arrow) of the aged AAT^–/– mice compared to the littermate controls or 2 M old mice of either genotype. (c) Immunofluorescence on retinal cross sections and western blot analysis of aged (18-24M) AAT^+/+ and AAT^–/– following a similar format as shown in (a). Expression of HTRA1 is increased (white arrows) at the BrM/RPE. HTRA1 overexpressing mice (HTRA1^OE, last panel) were used as a positive control for the HTRA1 antibody staining showing the overexpressed HTRA1 protein at the BrM/RPE region. Next to each panel is a higher magnification of the RPE and photoreceptor outer segment region (RPE/PS; Scale bar: 10 μm). Western blot analysis with RPE/choroid tissue extracts from AAT^+/+ (black bar) and AAT^–/– mice at 18-24 M of age confirming the changes seen by immunofluorescence is to the right. (a–c) All western blots used 6–7 retinas and stainings were confirmed in at least 3 different biological samples. Bars show percentage expression ± S.E.M.; *p<0.05; ***p<0.001 Scale bar: 50 μm except for high magnification where it is 10 μm; Blue: nuclear DAPI; green: ApoE in (a) and PNA to visualize cone segments in (b, c); red: CFH in (b) and HTRA1 in (c); BrM/RPE, Bruch’s membrane and retinal-pigmented epithelium layers; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer, vertical lines mark thickness of layers indicated.