The presence of lipofuscin, a highly oxidized insoluble protein that accumulates in the cytoplasm, was identified using Sudan Black B (SBB) and Fontana Masson staining only at 6 months of age. Pictures were quantified using ImageJ software, and a “stack image” and a color threshold were applied to identify the stained structure. Results are reported as a red stained area percentage among the total area [30 (link),42 (link)]. Additionally, the autofluorescence of liver lipofuscin was detected using an inverted fluorescent microscope (Eclipse Ti2 Series-Nikon) at birth and 6 months of age. Lipofuscin exhibits broad-spectrum autofluorescence [43 (link),44 (link)]. A GFP filter set was applied with an exposure time of 400 ms throughout the observation. Pictures were taken by a single examiner (C.Y.).
To localize lipofuscin deposits at 6 months of age, hepatocytes were stained with cytokeratin 18 (1/100, Abcam) overnight at 4 °C. Sections were then washed with PBS and incubated for two hours with Alexa Fluor-647-conjugated goat anti-rabbit IgG (1/200, Abcam). Sections were then rinsed with PBS and mounted using Fluoromount g mounting medium with DAPI. A negative control was established through incubation only with secondary antibody. Slides were observed blindly using a fluorescence microscope (Nikon, Eclipse Ti2 Series) by the same experimenter (C.Y.).
To localize lipofuscin deposits at 6 months of age, hepatocytes were stained with cytokeratin 18 (1/100, Abcam) overnight at 4 °C. Sections were then washed with PBS and incubated for two hours with Alexa Fluor-647-conjugated goat anti-rabbit IgG (1/200, Abcam). Sections were then rinsed with PBS and mounted using Fluoromount g mounting medium with DAPI. A negative control was established through incubation only with secondary antibody. Slides were observed blindly using a fluorescence microscope (Nikon, Eclipse Ti2 Series) by the same experimenter (C.Y.).
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