Figure 4.

Clearing time course and antibody penetration of PACT-processed samples. Quantitative comparison of the effect of different hydrogel-embedding conditions and clearing buffers on time to clear and antibody penetration during immunostaining. 1-mm-thick mouse coronal slices were hybridized and cleared with the array of previously used PACT conditions (Fig. 3). Slices were monitored for the time they took to become transparent. Once cleared, slices were washed and then immunostained. (a) Representative images of two 1-mm-thick coronal brain slices (~1.0–0.0 mm anterior to bregma¹⁴²) through the time course for PACT clearing and a comparison of time to clear (mean ± s.e.m.) for each PACT hydrogel composition. For the representative images, slices were cleared with 8% SDS-PBS (pH 8.5) and incubated in RIMS for 24 h. (b) Imaging of antibody penetration through different PACT tissue preparations. Previously cleared and washed 1-mm-thick slices were immunostained for parvalbumin (red) and nuclei stained with DAPI (cyan), using 2-d incubations with the primary and Fab format secondary antibodies (for immunostaining reagents, see Table 4), transferred to RIMS for 5 h and then RIMS-mounted. Samples from the cortex, traversing the depth of the slice, were imaged on a Zeiss LSM 780 confocal microscope with a Plan-Apochromat 10× 0.45 NA M27 air objective (w.d. 2.0 mm). To ensure even illumination throughout the depth of the slice for fair antibody detection, we applied laser power z-correction (Zen software, Zeiss): power was changed linearly for each slice, shown as a gradient next to each image; starting power values at the top were chosen to match the level of fluorescence at the surface across slices, whereas the range of powers varied for different PACT conditions. Shown are images of staining through A4P0, A4P1 and A4P4 hydrogel-embedded samples, as well as unhybridized tissue, cleared with 8% SDS-PBS (pH 7.5). As antibody and small-molecule dye diffused through both the top and bottom surfaces of the slice simultaneously, the images show that within 2 d DAPI has fully penetrated in all of the conditions, whereas antibody labeling has progressed to varying extents, depending on the PACT condition. As slices cleared with the different conditions also swell to different extents during the process (indicated by their difference in height relative to the pre-clearing height of 1 mm, as indicated by the white dotted lines in b), penetration of antibody through a more swollen sample will either require longer diffusion time or faster diffusion rate to reach an equivalent anatomical depth as in a less swollen sample. Incomplete detection of the DAPI signal in A4P1 and A4P4 slices is due to the difficulty of achieving similar light penetration in highly cross-linked slices. (c) Depiction of parvalbumin staining through same slices as in b. DAPI signal has been removed to better show the variable penetration of the antibody over the course of a 2-d period. (d) Quantification of antibody penetration through PACT conditions depicted in b and c. Antibody fluorescence signal was scaled by the average DAPI intensity for each z-section inside the volume and the average scaled fluorescence along a line perpendicular to the tissue surface produced a final estimate of labeling intensity (in arbitrary units, a.u.) as a function of tissue depth (Supplementary Methods). Antibody diffusion was fit to an exponential model [f(x) = a × exp (−tau × x) + b], with the exponent tau being inversely proportional to the square root of the diffusivity, wherein a larger tau indicates slower diffusion. Labeling intensities for A4P0, A4P1, A4P4 and unhybridized samples cleared with 8% SDS-PBS (pH 7.5), as a representative sample of all the different buffers, are plotted on a logarithmic scale. The amount of PFA contained in the hydrogel-tissue matrix is inversely proportional to immunohistochemical staining efficiency. Experiments on vertebrates conformed to all relevant governmental and institutional regulations, and they were approved by the Institutional Animal Care and Use Committee (IACUC) and by the Office of Laboratory Animal Resources at the California Institute of Technology.