“White and Clear” (WaC): A tunable hydrophilic tissue clearing strategy for multi‐species 3D imaging
Vicente Llorente, Elena Fernández‐Cortés, Daniel Sanderson, Julia Carolina Salazar Romero, Juan Miguel Gil‐Jaurena, Esther Bernaldo‐de‐Quirós, Rafael J. Jiménez‐Riobóo, Rafael Correa‐Rocha, Isabel Izquierdo‐Barba, Rafael Samaniego, Jorge Ripoll, Manuel Desco, María Victoria Gómez‐GaviroAbstract
Optical tissue clearing (OTC) techniques have transformed three‐dimensional (3D) biological imaging, yet existing protocols often face limitations related to pigment persistence, reagent viscosity, or restricted applicability across tissues and species. Here, we introduce White and Clear (WaC), a tunable hydrophilic tissue clearing strategy developed from the CUBIC (clear, unobstructed brain imaging cocktails) workflow that integrates efficient delipidation, hydrogen peroxide‐based pigment removal, and customizable refractive index matching using 2,2′‐thiodiethanol (TDE). WaC provides rapid, versatile, and reproducible clearing across multiple organs and species while preserving morphology and immunoreactivity, and enabling deep 3D imaging with confocal and light‐sheet microscopy.
WaC improves ease of use, versatility and clearing efficiency across samples from multiple species, including rodents, pigs, and humans; it also includes the capability for whole‐body clearing of small animals like mice; compatibility with 3D imaging techniques such as selective plane illumination microscopy (SPIM)/light‐sheet fluorescence microscopy (LSFM); the possibility of fine‐tuning clearing parameters for optimal results in different situations; and the avoidance of cumbersome synthesis or application procedures as well as the use of toxic reagents.