2.1. Construction of a Flow-Through Electrophoretic Cell
The electrophoretic cell was made from a 50 mL polyethylene (PE) vessel with a conical base and screw cap, which is normally used for collecting urine (P-LAB, Prague, Czechia). The sampling vessel was modified for use in the flow-through cell as follows (
Figure 1):
(i) Two holes with a diameter of 4 mm were drilled into the top of the conical base and the vertical wall cca 45 mm from the upper edge of the vessel. Plastic connectors for connecting rubes, a small Luer fitting for 1/8” tubing (the Luer valve assortment set, from World Precision Instruments, Saratosa, FL, USA, was used), were forced into the openings; the drilled hole must be slightly smaller than the diameter of the connector so that a water-tight connection is formed after pressing it in. The connectors were joined to transparent PE extension tubing for infusion probes with a length of cca 70 cm (Extension line, Braun, Melsungen, Germany). The other end of the lower tube is connected to the outlet stopcock of a 5 L stock bottle (P-LAB), once again using a fitting (World Precision Instruments, Saratosa, FL, USA), and is used to bring the washing solution to the cell. The other end of the upper tubing is loosely inserted into an opening in the screw top of a second 5 L stock bottle, which is used to collect solution flowing out of the cell.
(ii) The electrophoretic part of the cell consists of two platinum foils with a length of cca 2.1 cm, height of 1.0 cm, and thickness of 0.2 mm (Safina, Vestec, Czechia), acting as electrodes. The Pt-foils are inserted longitudinally into vertical slits cut with a scalpel in the lower part of the vessel (there is a space of cca 10 mm between the slits) so that 2 mm of the foil remains outside the vessel. Each slit is thoroughly sealed around its circumference with a special adhesive for PE (Ceys, AC Marca, Český Brod, Czechia), forming a mechanically strong and water-tight connection between the electrode and the outer surface of the vessel. Insulated copper wires are then soldered to the outside parts of the Pt-electrodes for connecting the DC voltage, see
Figure 2.
(iii) The hydrogel tissue is fixed inside the vessel using PE mesh with a hole size of 1–2 mm. The material for the mesh was window netting against insects, from which two circular pieces were cut; the first formed the bottom, on which the hydrogel tissue is placed, and the second is above the hydrogel and it is fixed in a position between the flat Pt-electrodes. It is important to use the meshes to keep the hydrogel between the electrodes at a place with high electric field intensity; otherwise, the hydrogel moves freely in the vessel under the effect of turbulent flow of the solution, substantially reducing the effectiveness of the electrophoretic washing and the hydrogel can be damaged mechanically.
2.2. Putting Together the Electrophoretic Washing Equipment
The electrophoretic flow-through cell is fixed in place using metal clamps and a chemical stand about 50 cm above the work surface of the fume cupboard. The input opening at the bottom of the cell is connected to PE tubing, bringing in the washing solution from the stock bottle located at a height of cca 100 cm above the work surface (
Figure 3). The washing solution flows through the cell under the force of gravitation from the bottom upwards at an average flow rate of 1 L/min, washes the surface of the hydrogel and flows out of the cell through the upper opening, from which it is fed through the other PE tubing to the lower stock bottle standing on the work surface. The solution is pumped from the lower to the upper vessel by a diaphragm pump (LS403, LILIE GmbH & Co. KG, Besigheim, Germany, pressure 2.1 bar, flow rate 10.6 L/min, 12 V charging voltage), originally designed for a garden irrigation system, which pumps the entire volume of 5 L in 0.5 min; the pump is connected to a 1/2” irrigation hose passing through the lids of the two vessels. Transfer of the washing solution is controlled automatically by two contact sensors located 5 cm under the upper edge of the stock bottles and proceeds as follows: When the surface of the solution in the bottom bottle reaches the position of the sensor, a switching relay is turned on and the pump begins to pump solution into the upper bottle. The pumping stops when the solution in the upper bottle reaches the position of the sensor, activating the switching relay and the pump is turned off. The whole process proceeds sequentially and was operated without any defect for a test period of 5 days.
The washing out of lipids from the hydrogel is accelerated by applying a DC voltage to the Pt-electrodes (DC Power Supply KXN-2005D, Zhaoxin, Shanghai, China). At an optimum value of the working voltage of 50 V, a current of about 1.1 A flows through the circuit and the temperature of the solution inside the cell does not exceed 42 °C, so that the hydrogel is not thermally degraded. The washing solution is electrolyzed at the Pt-electrodes, manifested in the evolution of bubbles of oxygen at the anode and of hydrogen at the cathode.
2.3. Tissue Samples, Fixation, and Preparation
Rat brains were obtained from adult male Wistar rats (10 months old) from the breeding colony (The Institute of Physiology, Academy of Sciences of CR). The rats were anaesthetized using 5%
m/
v pentobarbital intraperitoneally and perfused with standard physiological solution, then decapitated and their brains were extracted from their skulls and placed in a physiological solution for 24 hours. The cerebellum and brainstem were separated from the rest of the brain. The remaining brain tissue (part of telencephalon and diencephalon) was processed using the CLARITY system to form a transparent hydrogel, see
Figure 4. Human brains (4 males, aged 70–72 years, postmortem interval 4–6 days, without brain fixation in paraformaldehyde) were extracted from the skull of post mortem cadavers and dissected under a light magnifier (Fisher Scientific, Loughborough, UK). Nucleus accumbens was dissected from a coronal brain section as a cubic block specimen (5 × 5 mm) and treated by the CLARITY procedure.
2.4. Preparation of Solutions for CLARITY
All the chemicals were of analytical grade purity and all the solutions were prepared using deionized Milli-Q water (DEI, 18.2 MΩ cm, Millipore, Molsheim, France).
2.4.1. Components for Hydrogel Preparation
(i) the 40% m/v acrylamide (Amersham Biosciences, Uppsala, Sweden) solution was prepared in DEI and stored at 4 °C; (ii) 2% m/v bis-acrylamide (N,N-methylenebisacrylamide, Amersham Biosciences, Uppsala, Sweden) was prepared in DEI and stored at 4 °C; (iii) the phosphate-buffered saline stock solution (stock PBS) was prepared by dissolving 8.0 g NaCl (Lachema, Brno, Czechia), 0.2 g KCl (Lachema, Brno, Czechia), 1.44 g Na2HPO4 (Sigma-Aldrich, Steinheim, Germany), and 0.24 g KH2PO4 (Sigma-Aldrich, Steinheim, Germany) in 100 mL of DEI and the pH of the solutions was adjusted to 7.4 by 1 M HCl; iv) the 10% m/v paraformaldehyde (Lachema, Brno, Czechia) solution was dissolved in hot DEI and stored at 4 °C.
2.4.2. Penetration of Brain Tissue by the Hydrogel Solution
125 mg of VA-044 Initiator of polymerization (2,2-azobis (2-(2-imidazolin-2-yl) propane) dihydrochloride, Waco Chemicals, Neuss, Germany) was weighed into a 50 mL polyethylene tube and the following were added stepwise: 5 mL 40% m/v acrylamide solution, 1.25 mL 2% m/v bis-acrylamide solution, 5 mL 10 fold diluted stock PBS, and 20 mL 10% m/v paraformaldehyde solution. After mixing, the brain sample was immersed in the hydrogel solution and cold DEI was added to a final volume of 50 mL. The brain samples were incubated in the hydrogel solution at 4 °C for 2–3 weeks.
2.4.3. Hydrogel Tissue Embedding
Initially, one half of the hydrogel solution was removed before the acrylamide polymerization, which was performed in the same 50 mL PE flask used for sample penetration. First, the lid of the flask was replaced with a modified one equipped with a 4 mm hole and a male Luer fitting for attaching a laboratory vacuum pump (Julabo Laboport, Seelbach, Germany). The hydrogel solution was degassed under a vacuum of 0.9 bars (90 kPa) for 10 min. Following this procedure, the lid of the 50 mL flask was replaced with a common screw lid and the flask was placed in a water bath (Julabo TWB 5, Julabo Labortechnik, Seelbach, Germany) and incubated at 37 °C for 3 hours. Finally, the polymerized sample was removed from the flask, the excess gel was removed from the sample surface and the treated sample was inserted into an electrophoretic flow-through cell.
2.4.4. Preparation of the Clearing Solution
The clearing solution was prepared by dissolving: (i) 200 g of sodium dodecyl sulfate (SDS, Sigma-Aldrich, Steinheim, Germany) in 1.0 L of warm DEI and (ii) 62 g of boric acid (Sigma-Aldrich, Steinheim, Germany) in 4.0 L of DEI and the pH was adjusted to 8.5 with solid NaOH (Penta, Prague, Czechia). The two solutions were mixed in a storage bottle and used for electrophoretic clearing experiments.
2.5. Brain Tissue Staining and Confocal Microscopy
2.5.1. Brain Tissue Staining for Confocal Microscopy
CLARITY-processed brains were cut using a rat brain slicer (Adult Rat Brain Slicer Matrix BSRAS002-1, Pittsburgh, PA, USA) to 2 mm thick coronal slices. The brain slices were then washed with 0.1 M PBS, pH 7.4, for 24 hours. The specimens were incubated with primary antibody diluted in 0.1 M PBS and 0.3% m/v Triton-X with the addition of 0.01% m/v sodium azide for 7 days on an orbital shaker at room temperature (RT). The primary antibody was a rabbit polyclonal anti-Iba-1 (Wako, Osaka, Japan, 1:100) for rat brain. Immunohistochemistry of humans brain was carried out using 100 fold dilution of rabbit polyclonal anti-tyrosine hydroxylase antibody (TH, Millipore, Temecula, CA, USA, 1:100) and mouse monoclonal anti-parvalbumin antibody (PV, Sigma, St. Louis, MO, USA, 1:100). After incubation with the primary antibody, the sections were washed with 0.1 M PBS for 24 hours. Tissue slices were incubated with AlexaFluor 488 conjugated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, Baltimore, MD, USA) and AlexaFluor 594 conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch, Baltimore, MD, USA).. The secondary antibodies were 100 fold diluted in 0.1 M PBS and 0.3% m/v Triton-X and incubated on an orbital shaker at RT for 7 days. The tissue was then washed with 0.1 M PBS for 24 hours at RT to remove excess unbonded antibody.
2.5.2. Confocal Microscopy Procedure
Labelled brain slices were placed in a Cellvis Petri dish with 20 mm round bottom coverslips (
# 1.5) and supported by 0.1 M PBS. Microscopic imaging of the samples was performed with a high-end confocal laser scanning microscope Leica TCS SP8 X equipped with a white light laser (emitting in the spectral range 470–670 nm) and a near UV laser 405 nm, and two freely tunable spectral detectors: A PhotoMultiPlier and a hybrid detectors based on Gallium Arsenide Phosphide (GaAsP), see (
Figure 5 and
Figure 6). Leica objectives Plan Apo 10 ×/0.40 dry (working distance 2.2 mm) for low-resolution and Plan Apo 40/1, 10 Water (working distance 0.65 mm) for high-resolution were also used (
Figure 5 and
Figure 6). The attenuation of excitation laser power (and the associated emitted fluorescence signal) with increasing penetration depth was compensated by Acoustical Optical Tunable Filter settings of excitation lasers.
2.6. Ethical and Legal Statement
Human brains were obtained, on the basis of the informed consent from all the individual participants included in the study, for the Donor Program to the Third Faculty of Medicine, Charles University, Prague, CR and the study of human brains was approved by the Ethical Committee of the Third Faculty of Medicine (No. 12/2016). All the experimental procedures were approved by the Expert Committee for Protection of Experimental Animals of the Third Faculty of Medicine and were performed in accordance with the Animal Protection Act of the Czech Republic and respected the Guidelines of the Council of the European Union (86/609/EU).