HYBRiD: hydrogel-reinforced DISCO for clearing mammalian bodies

Animals

Mice were group-housed on a 12-hour light/dark cycle and fed a standard rodent chow diet. Room temperature was kept at 22 °C with humidity between 30–80% (not controlled). The following strains were purchased from The Jackson Laboratory for this study: wild-type C57BL/6J (stock no. 000664), B6.129P2-Pvalb^{tm1(cre)Arbr/J} (stock no. 017320, PV-Cre), B6.Cg-Sst^{tm2.1(cre)Zjh/J} (stock no. 013044, SST-Cre), and B6.Cg-Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze/J} (stock no. 007909, Ai9). PV-Cre and SST-Cre lines were crossed with Ai9 animals to generate the PV-Ai9 and SST-Ai9 animals used in this study. B6.Cg-Tg(K18-ACE2)2Prlmn/J (stock no. 034860, K18-hACE2) hemizygotes were used for SARS-CoV-2 infection experiments. B6.Cg-Tg(Thy1-EGFP)MJrs/J (stock no. 007788, Thy1-GFP-M) and B6.Cg-Tg(Thy1-YFP)HJrs/J (stock no. 003782, Thy1-YFP-H) animals were a gift from the Deisseroth lab at Stanford University. Both male and female mice were used for anatomical assays. All experimental protocols were approved by The Scripps Research Institute Institutional Animal Care and Use Committee (animal protocol 18-0001) and were in accordance with the guidelines from the US National Institutes of Health.

Sample collection

For brain, leg, and torso samples, adult PV-Ai9, Thy1-GFP, or Thy1-YFP mice (n = 4–6 per genotype) were heavily anesthetized with isoflurane and then transcardially perfused with ice-cold PBS followed by ice-cold 4% PFA in PBS with 4% sucrose (Electron Microscopy Perfusion Fixative, 1224SK). For juvenile whole-body clearing, littermates at 2 weeks (P12, n = 4) and 3 weeks (P21, n = 3) of age were prepared using the above procedures. For newborn whole-body clearing, PV-Ai9 or SST-Ai9 animals age P0–P3 (n = 4–6 per genotype) were anesthetized by hypothermia, and then the front of the rib cage was removed and animals were euthanized by puncture of the right atrium. All samples were washed with PBS and then placed in 4% PFA (in PBS) for post-fixation overnight at 4 °C. For SARS-CoV-2-infected mice, animals were euthanized at 5 days post infection, and their bodies were then submerged in zinc-formalin for fixation without perfusion at 4 °C for 3 days before transfer from the biosafety level 3 (BSL3) facility. After post-fixation, all samples were washed in 1× PBS at room temperature (RT) for 1 hour 3 times to remove residual PFA. All skin and hair was removed. Intact chest samples were prepared by removal of the chest from the body (diaphragm to cervical spine inside rib cage) using scissors, then the scapula and the brown adipose tissue were trimmed off. One sample from each group (wild type and K18-hACE2) was cut in half after clearing (along the central plane of the spine) to facilitate DAPI staining, while the rest were kept intact for fluorescent lightsheet microscopy.

Decalcification and decoloring

Samples (all except 1-mm brain slices, PEGASOS and SHANEL samples) were decalcified in 10% EDTA/15% imidazole solution (in water) with gentle shaking at 4 °C for 4 days¹⁰. Subsequently, the samples were washed in PBS with gentle shaking at RT overnight followed by 1-hour washes 3 times at RT. All samples were decolored using 25% N,N,N′,N′-tetrakis(2-hydroxypropyl)ethylenediamine (Quadrol) in 1× PBS with gentle shaking at 37 °C for 2 days (4 days for 3-week-old juveniles); solution was refreshed after 8 hours, then again at 24 hours. Intact SARS-CoV-2-infected chest samples were decolored for 6 days to aid in the removal of substantial amounts of pigment (heme) left in the tissues because they were not perfused. Subsequently, the samples were washed in PBS with gentle shaking at RT overnight followed by 1-hour washes 3 times at RT. Pretreated tissues were stored in 1× PBS with 0.02% sodium azide at 4 °C until clearing was completed.

CLARITY processing

CLARITY samples were first fixed with 4% PFA (in PBS) and then incubated in A1P4 hydrogel (1% acrylamide, 0.125% Bis, 4% PFA, 0.025% VA-044 initiator (w/v), in 1× PBS) at 4 °C for CLARITY embedding and subsequent passive clearing, as previously described¹⁵. Samples were transferred to hydrogel for 48–72 hours to allow monomer diffusion. The samples were degassed with nitrogen and polymerized (4 hours at 37 °C) with gentle agitation. Samples were removed from hydrogel and washed with 20 mM LiOH-Boric buffer, pH 8, containing 6% SDS at 37 °C overnight to remove residual PFA and monomers. Buffer was refreshed the next day, and every 3–4 days following during passive clearing. Passive clearing took place at 37 °C with continuous shaking until samples appeared translucent. After clearing, samples were washed in PBST (0.2% Triton X-100) for 2 hours, then the buffer was refreshed and samples were washed overnight at 37 °C to remove residual SDS. Cleared samples were RI matched (EasyIndex, RI = 1.52, LifeCanvas, or PROTOS²³) and incubated at 37 °C for 8 hours (up to 3 days) and then 6–8 hours at RT. A vacuum chamber was used to remove any air bubbles in the cleared samples before mounting and imaging.

iDISCO processing

We used a recent iteration of the iDISCO method, Adipoclear³, as previously described. All procedures were carried out at RT with shaking. Fixed samples were washed in 20%, 40%, 60%, and 80% methanol in H₂O/0.1% Triton X-100/0.3 M glycine (B1N buffer, pH 7), and 100% methanol for the wash times outlined below. Whole-mount samples were incubated in 100% methanol overnight at 4 °C before delipidation, with omission of the original peroxide bleaching step to preserve maximum fluorescence signal. Samples were then delipidated with 100% dichloromethane (DCM; Sigma-Aldrich), washed in 100% methanol twice, and then washed in 80%, 60%, 40%, and 20% methanol in B1N buffer. Samples were then washed in PBS/0.1% Triton X-100/0.05% Tween 20/2 mg/ml heparin (PTxwH buffer) for 1 hour 3 times, then overnight.

FDISCO processing

All procedures were carried out at 4 °C with shaking as previously described⁶. Fixed samples were washed in 50%, 70%, and 80% tetrahydrofuran (THF) in 25% quadrol (in 1× PBS to adjust to pH 9), then 95% THF twice. Timing of organic delipidation washes followed the outline given below, with 3-week-old juvenile whole body samples treated with dichloromethane 3 times. Samples were delipidated with 100% dichloromethane (DCM; Sigma-Aldrich) then washed in 95% THF twice, followed by 80%, 70%, and 50% THF. Samples were then washed in 0.2% PBST for 1 hour then 2 hours at RT, followed by 1× PBS for 1 hour 3 times then overnight at RT to thoroughly wash out any remaining organic solvent.

uDISCO, PEGASOS, and SHANEL processing

In comparison experiments, all procedures were completed as previously described, using passive protocols^5,22,24. For uDISCO, brain slices were incubated (for reflective index matching) with BABB-D4, and larger samples (legs and bodies) were incubated with BABB-D15, as suggested by the original paper. For PEGASOS, the hard-tissue protocol was used to clear limbs and newborn samples. For SHANEL, the protocol for passive clearing was followed, with modifications to timing due to the differences in sample size from the original publication, which was geared toward larger tissues. Timing of organic washes followed the outline given below.

Organic solvent protocol timing

Each organic clearing method (iDISCO, FDISCO, uDISCO, PEGASOS, SHANEL, HYBRiD) has provided a wide range of options for the length of the organic solvent washing steps according to sample types and fluorescence labels. To enable a direct and efficient comparison among them, we used a fixed wash duration (applied to both organic dehydration and delipidation) across all tested methods when processing the same type of tissues, as summarized here: 1-mm brain sections, 30 minutes; whole mount limbs, 45 minutes; newborn mouse (P0–P3), 90 minutes; adult mouse chest, 90 minutes; juvenile whole body, 180 minutes.

HYBRiD CLARITY embedding and additional clearing

HYBRiD samples were first processed with the rehydrated FDISCO procedure outlined above before gel embedding. See the CLARITY procedure outlined above. The intact chest samples contained high levels of excess blood, which required an increased duration of passive clearing to reduce. Chest samples were passively cleared for at least 28 days (up to 40 days). Active clearing using a SmartClear machine (LifeCanvas Technologies) could accelerate the clearing, but is not required. To remove all SDS from the tissue, samples were washed with 0.2% PBST at 37 °C with shaking twice for 2 hours then overnight, twice.

Immunostaining

Monoclonal rabbit anti-SARS-CoV nucleoprotein (Sinobiological, 40143-R019)^28,29 was directly conjugated to AlexaFluor 647 using the APEX Antibody Labeling Kit (Thermo Fisher Scientific, A10475) to allow single-step detection of viral infection in the cleared chest samples. Intact chest samples were incubated with the conjugated antibody at 37 °C for 4 days in 10% DMSO/0.2% PBST/0.02% sodium azide (1:700 dilution from the direct yield of APEX kit), then for an additional 4 days with an increased concentration (1:350 dilution) to maximize penetration of the antibody. Excess antibody was washed off with 0.2% PBST at 37 °C with shaking twice for 2 hours then overnight twice.

Viral infection

B6.Cg-Tg(K18-ACE2)2Prlmn/J (stock no. 034860, K18-hACE2) hemizygotes and wild-type C57BL/6J control mice were purchased from The Jackson Laboratory. Wild-type mice (n = 3) were used as mock-infected controls, and K18-hACE2 transgenic mice (n = 3) (6 mice in total) were infected for tissue-clearing experiments. All mice were maintained in parallel in a BSL3 facility for SARS-CoV-2 infection until euthanization. Mice were infected intranasally with 1 × 10⁵ PFU of SARS-CoV-2 in a final volume of 50 μl following isoflurane sedation. After viral infection, mice were monitored daily and euthanized at 5 days postinfection by CO₂. Overall timeline

Imaging

Light-sheet imaging

Whole body and intact chest images were acquired with a lightsheet microscope (SmartSPIM, LifeCanvas). Samples were mounted using 1% agarose/EasyIndex. Whole-body newborns were mounted with the dorsal side (spine) up, while intact chest samples were mounted dorsal side down (spine at the bottom of the Z-range). Samples were securely mounted to the holder after the mounting gel solidified (~20 minutes at 4 °C). Mounted samples were imaged inside the standard SmartSPIM chamber (90 × 65 × 45 mm) filled with 250 ml of EasyIndex after overnight equilibration. All the samples used in this study can be mounted using the standard chamber; however, for >2-week-old mice, only the chests can be visualized owing to the size limit of the standard chamber. To image the whole body of juvenile mice (2 and 3 weeks old), a custom oversized chamber (170 × 90 × 60 mm) was used and filled with 750 ml of EasyIndex. All images were acquired using a ×3.6, 0.2 NA objective (LifeCanvas), with z-step set to 4 µm, exposure time of 2 ms, and sampling at full resolution (2,048 × 2,048, 1.75/1.75/4 µm XYZ voxel size). Image acquisition was completed with bilateral illumination along the central plane of symmetry within the sample.

Confocal microscopy

Cleared samples were incubated in RI matching medium, then mounted to a glass microscope slide using spacers (1 mm, Sunjin Lab). Spacers were coated with silicone (KWIK-SIL, WPI) for use with samples in organic solvent to prevent degradation of the plastic. Tissues were then imaged with the Olympus FV3000 confocal microscope with one of the following objectives: ×4, 0.28 NA, air (XLFluor, Olympus); ×10, 0.6 NA, water immersion (XLUMPlanFI, Olympus); or ×40, 1.25 NA, silicone oil immersion (UPlanSApo, Olympus).

Image postprocessing

Raw data from the SmartSPIM were destriped and stitched using commercial software from LifeCanvas Technologies. Destriped and stitched images were converted to .ims format for 3D visualization in IMARIS (Bitplane, v9.2.1). Maximum intensity projections were captured as snapshots, and digital slice views were generated using the ortho slicer tool. Movies were captured using the recording feature in IMARIS. Confocal images were viewed and adjusted using Fiji (ImageJ v2.1.0).

Quantifications and image analysis

Transmittance

A 6-mm-diameter biopsy punch was used to cut 1-mm-thick brain slices to fit into the wells of a 96-well plate. For leg transparency measurements, 3-mm-thick cross section blocks were cut from the center of the upper hindleg to minimize tissue heterogeneity between samples; then, the punch was used to trim excess tissue. Samples were placed into wells with the minimum volume of RI matching medium needed to submerge them fully. Absorbance was measured by multiarea (5×5 grid across each well) at each wavelength 450 nm–75 nm with Cytation 3 plate reader (BioTek). The central 9 areas (3 × 3) of reading were used for quantification, as the edge of the wells gave varying results owing to the high viscosity of the RI matching medium. Blank was subtracted from raw absorbance values, then converted to transmittance (each condition n = 5).

Signal profile

Signal profile data were generated using Fiji. After opening a Z-projection (maximum intensity projection (MIP)) of an image, a straight line was drawn across an area including signal and background. The plot profile tool was used, and the signal plot data were recorded. Likewise, the same procedure was applied to areas of only background with no signal present. All signal profile plot data were normalized by dividing signal plot by the background plot values.

Tissue size measurement

Size measurement of linear changes were completed using brightfield images of 1-mm brain slices over a grid from each method in RI matching medium, subtracted from PFA-only slices in PBS. Calculations were completed using the square root of the area size change, as previously described⁶.

Fluorescence intensity and signal-to-background ratio

Fluorescence intensity and signal-to-background ratio were quantified using FIJI based on previous uDISCO and FDISCO publications^5,6. For each image, a maximum intensity projection over 50 μm of a region of interest (ROI) of 150–200 μm × 150–200 μm was generated on the surface of the tissue (within 150 μm from the top). The region of the signal was identified by auto-thresholding, and the rest of the image pixels were designated as region of background. For region of signal and region of background, total area, mean intensity, and sum of intensity were measured by FIJI. Fluorescence intensity was then calculated by ({mathrm{fluorescence}};{mathrm{intensity}} = frac{{{{{{mathrm{sum}}}}}left( {{{{{mathrm{signal}}}}}} right) – {{{{mathrm{mean}}}}}left( {{{{{mathrm{background}}}}}} right) times {{{{mathrm{area}}}}}({{{{mathrm{signal}}}}})}}{{{{{{mathrm{area}}}}}({{{{mathrm{signal}}}}})}}), and signal-to-background ratio was calculated by ({mathrm{Signal}};{mathrm{to}};{mathrm{background}};{mathrm{ratio}} = frac{{{{{{mathrm{mean}}}}}({{{{mathrm{signal}}}}})}}{{{{{{mathrm{mean}}}}}({{{{mathrm{background}}}}})}}). For each condition, 8–12 random ROIs in the cortex per tissue sample were quantified, and 4–6 tissue samples per condition were used in each analysis.

Protein loss

Samples treated with FDISCO and iDISCO protocols were evaluated for protein loss dependent on sequential hydrogel embedding in comparison to native and CLARITY embedded tissue with no pretreatment. Half-coronal 1 mm PV-Ai9 brain slices were briefly dabbed on a tissue paper to remove excess moisture before mass of tissue in each condition was recorded. All samples placed into LiOH-Boric SDS buffer following their respective treatments and were placed at 37 °C with shaking to begin passively clearing. On the fourth day of clearing, buffer was sampled from each tube, then fresh clearing buffer was added to each tube before samples were placed back on the 37 °C shaker. Samples continued passively clearing until day 7, when clearing buffer was once again collected. A Pierce BCA Protein Assay Kit (cat no. 23227, Thermo Fisher Scientific) was used for colorimetric quantification of protein content. Total protein loss values are the sum of day 4 and day 7 quantifications.

Statistics and reproducibility

Statistical significance was evaluated with Prism 8 (Graphpad) using t-tests and one- or two-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison post hoc test. All data presented as individual datapoint or mean ± s.e.m., with n values given in the figure legend as biological replicates.

All fluorescence intensity, transparency, size, and protein-loss experiments were repeated 3 times with n = 6. Newborn clearing experiments were repeated 3 times with n = 3 of each method, and each transgenic line. For juvenile clearing experiments, 3–4 biological replicates were compared at ages P12 (2 weeks) and at P21 (3 weeks); for SARS-CoV-2 samples, infection was performed once with 3 biological replicates in each genotype.

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.