Formalin-fixed paraffin embedded (FFPE) mouse brains were sectioned (8-μm thickness) and glass mounted. To reduce the autofluorescence signals by greater than 90% intensity (e.g., lipofuscin or hemosiderin), FFPE slides were photobleached up to 48 h using a multispectral LED array in the cold room overnight to reduce the autofluorescence in the brain tissue.75 (link) The sections were deparaffinized, PBS-washed and stained with 25 μM for 30 min. The sections were washed with PBS buffer and a coversliped with PermaFluor (Thermo) as the mounting media. For FFPE human brain sections, the same procedures were followed. For EMBER data collection, the same steps were taken without the autofocus function and with zoom of 1.5.
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Reducing Autofluorescence in FFPE Brain Tissue
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Corresponding Organization : University of California, San Francisco
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Variable analysis
independent variables
- Photobleaching duration (up to 48 h)
- Autofluorescence intensity reduction (greater than 90%)
- FFPE mouse brain sections (8-μm thickness)
- Glass-mounted FFPE sections
- Deparaffinization of sections
- PBS washing of sections
- Staining of sections with 25 μM for 30 min
- PBS washing of stained sections
- Mounting of sections with PermaFluor
- FFPE human brain sections (same procedures)
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