All the protocols adhered to the ARRIVE guidelines with the limited exception that antibody staining and clearing effectiveness was measured unblind. In these instances of unblinded data collection, the data analysis was conducted blind.
All use of vertebrate animals including animal care and experimentation was carried out in accordance with NIH and ARRIVE guidelines.
All procedures involving animals were approved by the Institutional Animal Care and Use Committee at the University of Massachusetts in Amherst (IACUC; protocol #2018-0014 and #2017-0060) and conducted in accordance with protocols approved by IACUC and in adherence with all relevant regulations and directives on animal care.
Euthanasia of mice was performed using isoflurane (2–5%) to induce deep anesthesia followed by cervical dislocation.
Measure of MHD-induced flow
A solution of sodium chloride was made in a small tank (2.5 L). Sodium chloride was slowly added to the tank until the electric conductivity of the solution matched that of the clearing solution. The clearing device was then submerged in the solution with a measured grid behind the tank to provide scale. 0V, 10V, 20V, 30V, 40V, 50V, or 60V were applied to the device and sodium alginate spheres were introduced into the tank at a constant location (N = 7). The velocity of the spheres through the device was measured. Velocity was calculated using a high-speed video taken over a calibrated grid. This process was then repeated using only an electric field (magnets were removed). Paired-sample t-tests were performed between the MHD and electric-only conditions at each voltage and a 2-way ANOVA was performed across all voltages using MATLAB. The p-values for the paired samples T-test were corrected for multiple comparisons using Bonferroni correction. Each condition was fit to a linear model using MATLAB.
Design of MHD-accelerated clearing device
The strategy for using MHD to remove lipids from tissue samples requires binding proteins and polymerizing a hydrogel, removing lipids, and matching the refractive index of the tissue and imaging media (Fig. 5A). A tissue chamber was placed into the central chamber of the MHD-accelerated clearing device (Fig. 5B, C). This holds the tissue at the intersection of the electrical and magnetic fields. The clearing chamber was submerged in a large (5 L) bath of clearing solution at 37°C and 30 VDC (0.35 Amps) was applied across the tissue for several hours (typically 16 hours for mouse brain tissue and 2 hours for intact zebrafish brains; Fig. 5D).
Tissue Fixation and hydrogel polymerization
Mice were anesthetized with isoflurane, euthanized, and perfused with 0.01 M phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in 0.01M PBS.
Tissue was then post-fixed in 4% PFA at 4 ˚C overnight. Next, the tissue was placed in a hydrogel solution (4% acrylamide, 4% PFA, 0.05% bis acrylamide, and 0.25% VA-044 initiator suspended in 0.01 M PBS) at 4 ˚C overnight (Chung, et al., 2013; Isogai, et al., 2017). Oxygen was flushed out of hydrogel-infused tissues nitrogen gas and then the samples were polymerized by incubating them at 37 ˚C overnight (Chung, et al., 2013). Excess hydrogel was removed from the surface and tissue samples were transferred to PBS to flush hydrogel monomers.
Adult zebrafish were euthanized in 0.2 mg/ml tricaine mesylate (MS-222), decapitated, and the heads placed in 4% paraformaldehyde overnight. Heads were then placed in PBS and brains were carefully dissected, incubated in hydrogel at 4 ˚C overnight, and processed as above.
Adult nudibranchs (Berghia stephanieae) were anaesthetized in cold 4.5% magnesium chloride in artificial sea water for 20 minutes, pinned to a Sylgard-lined dish, and fixed in 4% paraformaldehyde in sea water overnight at 4 ˚C. Whole animals were washed with PBS and then incubated in hydrogel at 4 ˚C overnight and processed as above.
Active Tissue Delipidation (clearing): Tissue samples were incubated in SDS-clearing solution (10 mM sodium dodecyl sulfate in 0.1 M borate buffer, pH 8.5) for 2 days at 37 ˚C unless otherwise noted. Samples were then transferred to the MHD-accelerated clearing chamber, consisting of two interlocking cell-strainers (ThermoFisher; catalog #: 87791). This chamber was placed in the intersection of the electrical and magnetic fields in the center of the device and the chamber was lowered into a bath of 37 ˚C SDS. 30V DC were then applied across the tissue to initiate MHD-accelerated clearing (Fig. 5D). After clearing, the tissue is taken out of the clearing chamber and washed in 0.1 M PBS for at least 12 hours. Of the 55 samples cleared for this paper using this technique in multiple laboratories, all achieved transparency with little physical damage.
Electrophorectic Clearing: Tissue samples were incubated in SDS-clearing solution for 2 days at 37 ˚C unless otherwise noted. Samples were then transferred to a clearing chamber, consisting of two interlocking cell-strainers (ThermoFisher; catalog #: 87791). This chamber was placed between two electrodes in the center of a MHD-accelerated clearing device, which has had magnets removed from the device. A 500ml/min peristaltic pump (Grey Beard Niagra) was then affixed to the top of the central chamber to circulate buffer across the tissue during clearing by pulling buffer from the temperature-controlled bath. The chamber and output from the pump were lowered into a bath of 37 ˚C SDS. Direct electrical current was then applied across the tissue to initiate clearing. After clearing, the tissue was taken out of the clearing chamber and washed in 0.1 M PBS for at least 12 hours.
Clearing Temperature Measurements: Tissue was left to incubate in SDS-clearing solution for 2 days at 37 ˚C, then allowed to cool to room temperature for at least 2 hours. Tissue was then subjected to either MHD-accelerated or electrophoretic clearing (n = 6) for 30 minutes with four different voltages applied across the tissue (30, 40, 50, and 60 VDC) in a 37 ˚C SDS bath. After clearing the tissue was rapidly removed from the device and imaged with an infrared thermal imaging camera (Hti-Xintai: HT-18) on a room temperature background. The highest observed temperature from each sample was recorded and the tissue was allowed to cool down to room temperature prior to additional experiments at different voltages.
Refractive Index Matching and Light Sheet Microscopy
The tissue was transferred to “Optiview” (Isogai, et al., 2017) refractive index matching solution and incubated at 37 ˚C for at least 12 hours to achieve optical clarity through RI matching (Fig. 5A; Isogai, et al., 2017). Samples were imaged at 5X or 20X magnification with a lightsheet microscope adapted for a 1.45 RI imaging solution (Zeiss Z1).
Measures of Clearing Efficacy
Tissue was left to incubate in SDS-clearing solution for 2 days at 37 ˚C. Tissue was then subjected to either MHD-accelerated or electrophoretic clearing (n = 6) for 24 hrs. Clearing was interrupted at 0hr, 6hr, 12hr, and 24hr. Tissue was washed with 0.01M PBS overnight, then equilibrated to RI 1.43 in Optiview (Isogai, et al., 2017) for at least two days at 37 ˚C. Tissue transparency was then measured by the percentage of light transmitted through the tissue suspended in an Optiview solution (Isogai, et al., 2017). Light transmission was measured using a wide-spectrum light-source and calibrated photodiode. The sample was then washed in 0.01M PBS overnight, then equilibrated to SDS-clearing solution for 2 days at 37 ˚C before clearing continued up to 24hr per sample. Data across all samples at each time were fit with a saturating exponential curve in MATLAB.
MHD-accelerated staining of fixed tissue with methylene blue
Penetration of methylene blue into a 1 cm3 cube of homogeneous brain tissue under MHD force was tested over 1, 2, and 4 hours (N = 1). Cubes of uncleared sheep brain tissue were equilibrated to the antibody labeling buffer solution for 12 hours. The tissue was then placed at the intersection of a strong magnetic and electric field (30V DC) and submerged in a solution of methylene blue (0.1 M) buffered to pH 9.5 (37°C). The orientation of the electric field was reversed at 15-minute intervals for 3 minutes. Three samples were labeled using this approach for 1, 2 or 4 hours. Following the stain, the tissue was bisected and imaged. A control sample was incubated in the same solution (37°C) for 4 hours without the application of any active force. This sample was bisected and imaged as the others.
Comparative staining of methylene blue into agarose cubes as a result of various strengths of electrical force conjugated to MHD force
15 1 cm3 of 3 % agarose were subjected to labeling methylene blue labeling by MHD force for 0, 5, 10, 15, 30, 60, or 120 minutes at varied electrical field strengths. The distance penetrated into the agarose cubes was measure after bisection and plotted against staining time with 10, 20, or 30V in a constant magnetic field.
Antibody Labeling: Delipidated tissue was placed inside of a 2-inch length of 0.25-inch diameter dialysis tubing (6–8 kDa); Spectrum). After equilibration, samples were incubated in an antibody solution inside dialysis tubing at the center of intersecting electrical and magnetic fields where the MHD force was strongest (Fig. 6). Confining the tissue sample inside dialysis tubing reduced the volume of antibody required for labeling and protected the tissue sample and antibody solution from direct exposure to the electrodes. Magnets (Applied magnets; NB057-6-N52) were placed on the top and bottom of the MHD labeling device creating a central chamber Fig. 6B). The ends of the dialysis tubing were connected to 9.5 mm diameter vinyl tubing (ThermoFisher: S504591) using 0.25-inch Leur lock barbs (Cole-Parmer; UX-45501-20) to create a torus-shaped chamber allowing the antibody solution to circulate continuously and provide an even and continuous source of antibody to the tissue sample (Fig. 6). Antibody solution (4.5 mL; 0.1 M borate buffer titrated to pH 9.5 with 0.1 M LiOH, 1% heparin, 0.1% Triton X-100; 1:500 primary antibody) was transferred into the dialysis tubing using a 5 mL syringe. The labeling chamber was submerged in a 1L tub containing electrophoresis solution (0.1M Borate Buffer pH 9.5/0.1% Triton X-100 solution). A 5 mL syringe filled with the buffer solution was attached to the circulation line to maintain constant pressure inside of the dialysis tube. 60 volts DC (~ 0.2 Amps) was applied across the electrodes for 15 minutes, followed by 3 minutes of inactivity repeatedly for 12 hours to drive antibodies into the tissue sample. The system was held at 37 ˚C and protected from ambient light to minimize bleaching of fluorophores throughout the procedure.
Following each round of MHD-accelerated labeling, the antibody solution was replaced with a wash solution (0.1 M borate buffer titrated to pH 9.5 with 0.1 M LiOH, 1% heparin, 0.1% Triton X-100) and the tissue was exposed to 6-hours of “active washing” using the same voltage settings. Labeled tissue was then washed in 0.01 M PBS for at least 12 hours.
Traditional Immunohistochemistry: Mouse brains were dissected from highly anesthetized mice. These tissues were incubated in 4% PFA suspended in 0.01M PBS at 4°C. Tissue was sliced to 100 µm thickness on a vibratome and transferred to 0.01M PBS or the electrophoresis buffer used in MHD-accelerated labeling. Slices were blocked in 10% FBS in 0.5% TritonX-100/PBS or electrophoresis buffer at room temperature for 1 hour, then incubated in a 1:200 dilution of antibody in 10% FBS/PBS or electrophoresis buffer at room temperature for 2 hours. The tissue was then washed three times in 0.05% TritonX-100 / PBS or electrophoresis buffer for 30 minutes at room temperature.