Computational protocol: Lack of Neuronal IFN-β-IFNAR Causes Lewy Body- and Parkinson’s Disease-like Dementia

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Protocol publication

[…] For immunohistochemistry (IHC) and immunofluorescence (IF), either mice were perfused and brains fixed in 4% paraformaldehyde (PFA) and paraffin embedded or brains were dissected and snap-frozen before sectioning. In vitro neuronal cultures were fixed in 4% PFA before staining. Tissues and cells were stained as described ().For neurogenesis and BrdU staining, mice were injected intraperitoneally (i.p.) once per day (75 μg/g body weight) for 5 consecutive days and sacrificed 2 hr after last injection. Brains were processed and immunostained for BrdU and doublecortin (DCX).MMP was measured by adding tetramethylrhodamine ethyl ester (TMRE) and Hoechst (Life Technologies) to primary CNs.IF images were taken with a Zeiss LSM510 confocal scanning microscope and IN Cell Analyzer 2200 automated microscope. IHC images were taken with a NanoZoomer 2.0-HT digital slide scanner or Olympus BX51 microscope. Images were quantified with ImageJ (Fiji version), IN Cell Investigator, CellProfiler, Zeiss Zen, and Adobe Photoshop.In situ apoptosis detection was with TUNEL kits (Calbiochem) with Hoechst counterstaining or DAB substrate and methyl green counterstaining kits (R&D Systems).For transmission electron microscopy (TEM), 12-month-old mice were cardiac perfused with 2% PFA and 0.2% glutaraldehyde and paratenial, and central medial thalamic nuclei were dissected and processed for epon embedding and ultrathin sectioning. Samples were incubated with primary α-syn antibodies (Leica) and biotinylated (Vector Laboratories) or 1.4-nm gold-labeled secondary antibodies (Nanoprobes) and embedded in epoxy resin. Ultra-thin sections were analyzed in a Philips CM100 electron microscope. […]

Pipeline specifications

Software tools ImageJ, CellProfiler
Applications cryo-EM, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Dementia, Parkinson Disease, Neurodegenerative Diseases
Chemicals Dopamine