Computational protocol: Melanopsin retinal ganglion cell loss in Alzheimer disease

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Protocol publication

[…] Flat‐mounted retinas from 3 controls and 4 age‐matched AD patients, all belonging to the same postmortem cohort, were treated by antigen retrieval solution (ChemMate; Dako, Carpinteria, CA; code No. S2367 in distilled water, pH 9) at 80 °C for 1½ to 2 hours before processing for IHC using the antimelanopsin antibody (code No. 5J68). IHC detection for melanopsin on flat‐mounted retinas was performed as previously described., Briefly, after incubation of the primary antibodies for 72 to 84 hours at 4 °C (diluted 1:10,000), melanopsin was visualized using the Dako Envision kit (code No. K4002, diluted 1:2) and tyramide‐conjugated Alexa 488 (Molecular Probes, Eugene, OR, USA). Images were obtained using an iMIC confocal microscope (FEI, Till Photonics, Munich, Germany) equipped with appropriate filter settings for detecting 4′,6‐diamidino‐2‐phenylindole (DAPI) and CY2/Alexa 488. For a more detailed visualization of the melanopsin immunoreactive network of outer and inner stratifying dendritic processes, Z‐stacks with a focal depth of 30 to 40 μm were generated and the Z‐stacks of images were stitched using the LA‐stitch module of Fiji/ImageJ, version 1.47n, 64 bit. Images for 3‐dimensional (3D) analysis were obtained by the spinning disk confocal part of the microscope. Z‐stacks of typically 60 to 70 images separated at the Z‐level by 0.5 µm were deconvoluted in AutoQuant X, version 3.02 (Media Cybernetics, Rockville, MD, USA), and the localization of dendritic processes and cell bodies was further analyzed by IMARIS, version 7.6.4 (Bitplane Scientific Software, Zurich, Switzerland) using the filament tracer module. After deconvolution, an autoNetwork was created for each cell analyzed. Thereafter, the lowest (L) and highest (H) values of dendritic diameters were defined (L = 0.323 µm, H = 16.15 µm). We identified dendrites belonging to the analyzed cells by manual tracer, the measures obtained were exported into a spreadsheet, and values from each cell were computed as a mean value. […]

Pipeline specifications

Software tools ImageJ, AutoQuant, Imaris
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Alzheimer Disease, Liver Diseases, Optic Nerve Diseases, Retinitis