Light-Sheet Fluorescence Microscopy: Chemical Clearing and Labeling Protocols for Ultramicroscopy.
Light-sheet microscopy is an effective technique in neuroscience, developmental biology, and cancer research for visualizing and analyzing cellular networks and whole organs in three dimensions. Because this technique requires specimens to be translucent they commonly have to be cleared before microscopy inspection. Here, we provide 3DISCO based protocols for preparing cleared samples of immuno-stained neural networks, lectin-labeled vascular networks, and Methoxy-X04 labeled beta-amyloid plaques in mice. 3DISCO utilizes the lipophilic solvents tetrahydrofuran (THF) and dibenzylether (DBE) for dehydration and successive clearing. Crucial steps for obtaining transparent tissues and preserving the fragile endogenous GFP are the transcardial perfusion, as well as the proper implementation of the 3DISCO clearing process using peroxide free chemicals. We further provide a protocol for resin embedding of 3DISCO cleared specimens that allows long term archiving of samples for years with virtually no loss in signal quality.
Department of Bioelectronics, FKE, Vienna University of Technology, Floragasse 7, 1040, Vienna, Austria. nina.jaehrling@tuwien.ac.at.|Center of Brain Research, Medical University of Vienna, Vienna, Austria. nina.jaehrling@tuwien.ac.at.
Nina
N
Jährling
Department of Bioelectronics, FKE, Vienna University of Technology, Floragasse 7, 1040, Vienna, Austria.|Center of Brain Research, Medical University of Vienna, Vienna, Austria.
Klaus
K
Becker
Department of Bioelectronics, FKE, Vienna University of Technology, Floragasse 7, 1040, Vienna, Austria.|Center of Brain Research, Medical University of Vienna, Vienna, Austria.
Saiedeh
S
Saghafi
Department of Bioelectronics, FKE, Vienna University of Technology, Floragasse 7, 1040, Vienna, Austria.|Center of Brain Research, Medical University of Vienna, Vienna, Austria.
Hans-Ulrich
HU
Dodt
D000818:Animals; D001921:Brain; D021621:Imaging, Three-Dimensional; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D013116:Spinal Cord
3D reconstruction; 3DISCO; Alzheimer disease; Chemical clearing; Light-sheet microscopy; Peroxide elimination; Vascular networks; Whole-mount immune-labeling