Figure 1

Optimized immunofluorescence procedure on human brain with tissue quality screening. (A) Quenching of lipofuscin autofluorescence signal in human brain sections with TrueBlack treatment. Confocal imaging of inferior parietal layer V pyramidal cortical neurons immunostained with MAP2 antibody are shown on the far left panels. The two central panels illustrate the autofluorescence of lipofuscin in the red and far-red channels. The far right panels show the merged images. The top panel illustrates results in the absence of TrueBlack treatment and shows prominent lipofuscin autofluorescence in the red and far red channels and in the merged image. The bottom panel shows complete quenching of the lipofuscin signal with TrueBlack treatment. The images were taken under 63x objective lens. Scale bars 5 µm. (B) Quenching of nonspecific binding of anti-rabbit IgG secondary antibody. Tissues from brains with AD changes had marked non-specific binding of anti-rabbit IgG which can be prevented by 0.5% Tween20 in secondary antibody incubation buffer. The top panel shows section of parietal cortex from a control subject. The bottom panel corresponds to parietal cortex from an AD case. On the left, in the absence of surfactant there is prominent non-specific binding of anti-rabbit IgG, which disappears in the presence of 0.5% Tween20. The images were taken under 20x objective lens. Scale bars 20 µm. (C and D) Purkinje cells of the cerebellum immunostained with Beta-tubulin antibody. In (C), the left panel shows the structural integrity of the cell body and dendrites in a high-quality tissue sample. In contrast, the right panel shows the loss of immunostaining and disintegration of dendrites in a low-quality tissue sample. The images were taken under 20x objective lens. Scale bars 20 µm. In (D), the left panel shows well preserved axonal morphology and the right panel shows fragmentation of axons. The images were taken under 10x objective lens. Scale bars 50 µm. (E) Neurons of the parietal cortex immunostained with MAP2 antibody. The left panel shows well preserved cell bodies and dendrites. The right panel demonstrates fragmentation of dendrites and deformed cell bodies. The images were taken under 20x objective lens. Scale bars 20 µm. (F) Parietal cortex white matter astrocytes immunostained for GFAP. Left panel shows intact cell bodies and processes, which are disintegrating in the right panel. The images were taken under 20x objective lens. Scale bars 20 µm. (G) Tissue quality versus postmortem interval. Postmortem tissues (n = 36) were classified as high- or low-quality based on the integrity of Purkinje cells as described in (C). The plots show that there is no correlation between postmortem interval and tissue preservation (p = 0.5907). The bars in the graph represents the mean of postmortem interval in each group. (H) Demography and neuropathological diagnoses of autopsied subjects assessed in (G).