Logan A Campbell1, Katy E Pannoni1, Niesha A Savory2, Dinesh Lal3, Shannon Farris4. 1. Fralin Biomedical Research Institute, Center for Neurobiology Research, Virginia Tech Carilion, Roanoke, VA, USA. 2. Fralin Biomedical Research Institute, Center for Neurobiology Research, Virginia Tech Carilion, Roanoke, VA, USA; School of Neuroscience, Virginia Tech, Blacksburg, VA, USA. 3. Virginia Tech Carilion School of Medicine, Roanoke, VA, USA. 4. Fralin Biomedical Research Institute, Center for Neurobiology Research, Virginia Tech Carilion, Roanoke, VA, USA; Virginia Tech Carilion School of Medicine, Roanoke, VA, USA; Department of Biomedical Sciences & Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA. Electronic address: Farrissl@vtc.vt.edu.
Abstract
BACKGROUND: Protein expansion microscopy (proExM) is a powerful technique that crosslinks proteins to a swellable hydrogel to physically expand and optically clear biological samples. The resulting increased resolution (~70 nm) and physical separation of labeled proteins make it an attractive tool for studying the localization of subcellular organelles in densely packed tissues, such as the brain. However, the digestion and expansion process greatly reduce fluorescence signals making it necessary to optimize ExM conditions per sample for specific end goals. NEW METHOD: Here we compare the staining and digestion conditions of existing proExM workflows to identify the optimal protocol for visualizing subcellular organelles (mitochondria and the Golgi apparatus) within reporter-labeled neurons in fixed mouse brain tissue. RESULTS: We found that immunostaining before proExM and using a proteinase K based digestion for 8 h consistently resulted in robust fluorescence retention for immunolabeled subcellular organelles and genetically-encoded reporters. COMPARISON WITH EXISTING METHODS: With these methods, we more accurately quantified mitochondria size and number and better visualized Golgi ultrastructure in individual CA2 neurons in the mouse hippocampus. CONCLUSIONS: This organelle optimized proExM protocol will be broadly useful for investigators interested in visualizing the spatial distribution of immunolabeled subcellular organelles in various reporter mouse lines, reducing effort, time and resources on the optimization process.
BACKGROUND: Protein expansion microscopy (proExM) is a powerful technique that crosslinks proteins to a swellable hydrogel to physically expand and optically clear biological samples. The resulting increased resolution (~70 nm) and physical separation of labeled proteins make it an attractive tool for studying the localization of subcellular organelles in densely packed tissues, such as the brain. However, the digestion and expansion process greatly reduce fluorescence signals making it necessary to optimize ExM conditions per sample for specific end goals. NEW METHOD: Here we compare the staining and digestion conditions of existing proExM workflows to identify the optimal protocol for visualizing subcellular organelles (mitochondria and the Golgi apparatus) within reporter-labeled neurons in fixed mouse brain tissue. RESULTS: We found that immunostaining before proExM and using a proteinase K based digestion for 8 h consistently resulted in robust fluorescence retention for immunolabeled subcellular organelles and genetically-encoded reporters. COMPARISON WITH EXISTING METHODS: With these methods, we more accurately quantified mitochondria size and number and better visualized Golgi ultrastructure in individual CA2 neurons in the mouse hippocampus. CONCLUSIONS: This organelle optimized proExM protocol will be broadly useful for investigators interested in visualizing the spatial distribution of immunolabeled subcellular organelles in various reporter mouse lines, reducing effort, time and resources on the optimization process.
Authors: Denis P Shamonin; Esther E Bron; Boudewijn P F Lelieveldt; Marion Smits; Stefan Klein; Marius Staring Journal: Front Neuroinform Date: 2014-01-16 Impact factor: 4.081
Authors: Georgia M Alexander; Logan Y Brown; Shannon Farris; Daniel Lustberg; Caroline Pantazis; Bernd Gloss; Nicholas W Plummer; Patricia Jensen; Serena M Dudek Journal: Elife Date: 2018-11-02 Impact factor: 8.140
Authors: Fabian U Zwettler; Sebastian Reinhard; Davide Gambarotto; Toby D M Bell; Virginie Hamel; Paul Guichard; Markus Sauer Journal: Nat Commun Date: 2020-07-07 Impact factor: 14.919
Authors: Daniel Normen Düring; Mariana Diales Rocha; Falk Dittrich; Manfred Gahr; Richard Hans Robert Hahnloser Journal: Front Neuroanat Date: 2019-01-31 Impact factor: 3.856