Fig. 2

Histological and molecular changes in AAT^–/– mice. (a) Immunofluorescence on retinal cross sections of 18-24 M old wildtype mice (AAT^+/+, left column) and AAT^–/– mice (right column). Collagen IV (COL IV, red; first row) shows a reduction in signal at the RPE/BrM, while the laminin 3 (green; second row) and laminin 1,2 (red; third row) signal is increased. Scale bar: 50 μm; Blue: nuclear DAPI; red: Phalloidin in second row marking RPE cells; green: PNA in third row marking photoreceptor cells; 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; white arrows mark RPE/BrM region. (b) Western blot analysis with RPE/choroid protein extracts from 18-24 M old mice of genotypes indicated. Elastin levels are reduced while Laminin 3 levels are increased in AAT^–/– mice, confirming the histological findings in (a). Examples of western blot bands are shown beneath the bar graph. Bars show percentage protein ± S.E.M.; N = 4–5 mice; **p < 0.01; ****p < 0.0001. (c) Clinical grading of histological immunofluorescence stainings shown in panel (a) with mice 18-24 M of age. Antibody staining for the proteins in question was analyzed across the entire section and a percentage number was assigned as to the uniform covered length of the signal across the entire section. Each dot represents the eye of 1 animal. (d) Immunofluorescence on retinal cross sections of 2 M old wildtype mice (AAT^+/+, left column) and AAT^–/– mice (right column). Same antibodies and labeling were used as shown in panel (a), with no obvious change in expression seen at the RPE/BrM for all these proteins.