EZ Clear for simple, rapid, and robust mouse whole organ clearing

Abstract

Tissue clearing for whole organ cell profiling has revolutionized biology and imaging for exploration of organs in three-dimensional space without compromising tissue architecture. But complicated, laborious procedures, or expensive equipment, as well as the use of hazardous, organic solvents prevent the widespread adoption of these methods. Here, we report a simple and rapid tissue clearing method, EZ Clear, that can clear whole adult mouse organs in 48 hr in just three simple steps. Samples stay at room temperature and remain hydrated throughout the clearing process, preserving endogenous and synthetic fluorescence, without altering sample size. After wholemount clearing and imaging, samples processed with EZ Clear can be subjected to downstream applications, such as tissue embedding and cryosectioning followed by standard histology or immunofluorescent staining without loss of fluorescence signal from endogenous or synthetic reporters. Furthermore, we demonstrate that wholemount adult mouse brains processed with EZ Clear can be successfully immunolabeled for fluorescent imaging while still retaining signal from endogenous fluorescent reporters. Overall, the simplicity, speed, and flexibility of EZ Clear make it easy to adapt and implement in diverse imaging modalities in biomedical research.

Whole-Body and Whole-Organ 3D Imaging of Hypoxia Using an Activatable Covalent Fluorescent Probe Compatible with Tissue Clearing

“…Furthermore, we conducted simultaneous staining of hypoxia and blood vessels in the kidney. It has been reported that major renal blood vessels and partial glomerular capillaries can be labeled with Evans blue. , We prepared a kidney sample in which blood vessels were labeled with Evans blue and in which hypoxic regions were labeled with Pimo-BODIPY. By fluorescence imaging with LSFM, we succeeded in the simultaneous 3D visualization of renal hypoxia (green in Figure E) and blood vessels (red in Figure E) (Movie 2).…”

IgG hexamers initiate acute lung injury

“…After washing, Cy3 or Alexa Fluor 647-conjugated cross-adsorbed polyclonal secondary antibodies targeting rat, rabbit, goat and/or mouse IgG (Jackson Immunoresearch Cat# 712-165-153, Cat# 711-165-152, Cat# 705-605-147 and/or Cat# 115-605-206) were incubated with sections at 1:500 in PBS + 0.3% triton X-100 overnight. After additional washes, sections were either mounted in Vectashield (Vector Laboratories Cat# H-1700) for standard confocal imaging on a Nikon A1r microscope, or cleared after staining using the EZ clear protocol (47) for 3D imaging with a Nikon AZ100M confocal microscope.…”

An injury-induced tissue niche shaped by mesenchymal plasticity coordinates the regenerative and disease response in the lung

“…Lungs were fixed overnight at 4oC with gentle agitation. Lungs were then cleared using the EZclear protocol( 44 ). Briefly, the next morning, lungs were washed 4x 30 minutes each in PBS then incubated with 50% (v/v) THF (Sigma-Aldrich) in sterile milli-q water for 16 hours.…”