Figure 1
Development of enhanced CLARITY methods for peripheral tissues. (a) Simplified CLARITY schematic: First, fixed tissue is placed in a hydrogel monomer solution (purple) that diffuses throughout the tissue. Second, prior to polymerization, samples are transferred to a second hydrogel solution without the capacity for solid polymer formation (orange). Third, the sample is polymerized either through nitrogen flush and degassing or with the use of oil on the surface (indicated in yellow) as an oxygen inhibitor, resulting in formation of a tissue-polymer hybrid with a solid hydrogel interior but in liquid solution, which is ideal for fragile or irregularly shaped tissues. Fourth, tissue is passively cleared. Finally, after optical transparency is achieved, tissue is labeled with molecular probes (RNA, antibody, or dye), mounted on a slide in a refractive index matching solution, and imaged using standard microscopy. The sample can be destained and the process repeated for multiple staining and imaging cycles. (b) Virtual thin section (5 μm) compared to CLARITY volume (250 μm maximum intensity projection) of the same pancreatic islet image, here from a representative islet in a whole P15 pancreas from a Wnt1-Cre; ROSA-mTmG mouse. CLARITY enables more comprehensive and accurate measurements of structural features compared to section histology. Thin sections can grossly underestimate pancreatic islet sizes (dotted line), and results in incomplete observation of fine microstructures such as complex neural projections (arrowheads). (c) CLARITY gel A4B4P0 (green) clears significantly more rapidly compared to both A1B1P4 (red), and A4B4P4 (blue) across multiple tissue types including brain, muscle, liver, kidney (p < 0.00005, unpaired t-test), and pancreas (p < 0.0005, unpaired t-test). n = 6 sections per tissue type and gel. Clearing reaction rates were determined by fitting UV-spectrophotometry measurements over time to a first order transformation reaction kinetics equation y = Ymax(1 − e−kt) and normalized to Brain A4B4P4. (d) Different CLARITY gel formulations do not impact the loss of protein in a cleared specimen compared to PFA-alone fixed tissue, as determined by BCA assay (p < 0.00005, unpaired t-test). n = 6 samples per tissue type and gel. (e) The A1B1P4 formulation improves the penetration rate of antibodies, resulting in full 1 mm brain slices stained with 12hrs incubation in antibody, visualized using the marker Parvalbumin (PV). Scale bar = 100 μm. (f) Staining quality from (e) was quantified by measuring average cellular signal intensity normalized by average extracellular background intensity. n = 6 samples per gel type. For all charts, error is s.e.m. **p < 0.005, ***p < 0.0005, ****p < 0.00005.
