Fume Hood

Steps 1–7 need to be done in a fume hood.

Hydrogel solution preparation: (1) Combine and mix 40 ml of acrylamide (40%), 10 ml of bis-acrylamide (2%), 1g of VA-044 initiator (10% wt), 40 ml of ×10 PBS, 100 ml of 16% PFA and 210 ml of dH₂O with special attention to temperature and safety precautions. Keep all components on ice at all times to prevent polymerization. Caution: PFA is an irritant, sensitizer, carcinogen and toxin. Acrylamide is a potent neurotoxin, a respiratory and skin sensitizer, carcinogen, irritant, mutagen, teratogen and reproductive hazard. Many of the chemical constituents of hydrogels that could be used for CLARITY would fall into one or more of these categories. Therefore, to avoid skin contact or inhalation of monomers and/or crosslinkers (for example, acrylamide or PFA), solution preparation and all subsequent handling of hydrogel solution and polymer must be conducted in a fume hood with personal protective equipment including face shield, laboratory coat, gloves and closed-toe shoes.
Saponin is a widely used mild non-ionic surfactant often used to permeabilize cellular membranes in conventional immunohistochemistry. In CLARITY, saponin can be used in the hydrogel monomer infusion process to facilitate diffusion of the hydrogel monomer and initiator into the tissue, particularly for samples in which cardiac perfusion is not feasible, such as post-mortem human tissues and zebrafish brains. Saponin shortens incubation time required in the hydrogel monomer infusion process. However, bubbles may form that could be linked to saponin use, so routine saponin is not suggested. (2) Distribute 40-ml aliquots into 50-ml conical tubes on ice. Each tissue sample will require the use of one 40-ml tube: 20 ml for perfusion and the remaining 20 ml for sample embedding. (3) Seal tubes tightly and keep in secondary containment (on ice) before removing them from the hood. Transfer aliquots from ice to −20 °C. Store these solutions at −20 °C until they are ready to be used. They can be stored at −20 °C indefinitely if all components were kept sufficiently cold during the preparation process.
Solution preparation: (1) Combine and mix 123.66 g boric acid, 400 g sodium dodecyl sulphate, and 9 l dH₂O. To avoid skin contact or inhalation, prepare solution in a fume hood in proper PPE. Paying special attention to safety, combine water, boric acid and SDS while stirring. Add dH₂O to 10 l and add NaOH until the pH has reached 8.5. This solution can be made, stored and used at room temperature (20 °C). Caution: SDS is a toxin and irritant to the skin and respiratory system.
Transcardial perfusion with hydrogel solution: (1) Before perfusing, thaw the frozen hydrogel monomer solution in the refrigerator or on ice. (2) When the solution is completely thawed and transparent (but still ice cold), gently invert to mix. Make sure that there is no precipitate and avoid introducing any bubbles into the solution. (3) Prepare perfusion materials within a fume hood. (4) Deeply anaesthetize adult mouse with Beuthanasia-D. (5) Perfuse the animal transcardially, first with 20 ml of ice-cold 1× PBS and then 20 ml of the ice-cold hydrogel solution. Maintain a slow rate of perfusion (about 2 min for the 20 ml of each solution). (6) Immediately place the tissue (for example, brain) in 20 ml cold hydrogel solution in a 50-ml conical tube. Keep the sample in solution on ice until it can be moved to a 4 °C refrigerator. (7) Cover sample in aluminium foil if it contains fluorophores and incubate at 4 °C for 2–3 days to allow for further diffusion of the hydrogel solution into the tissue.
Hydrogel tissue embedding: (1) De-gas the 50-ml conical tube containing the sample in the desiccation chamber (in a fume hood) to replace all of the gas in the tube with nitrogen (as oxygen impedes hydrogel formation), as follows: place the 50-ml conical tube on a rack in the desiccation chamber; twist the 50-ml conical tube open sufficiently to allow gas exchange; turn on the nitrogen tank and adjust the control valve such that the inlet to the desiccation chamber fills with nitrogen gas; switch the desiccation chamber valve from nitrogen gas flow to the vacuum. Turn on the vacuum pump; verify that the chamber is under full vacuum by testing the chamber lid. Keep vacuum on for 10 min; switch the vacuum off and slowly turn the valve to fill the chamber with nitrogen gas; carefully open the chamber just enough to reach the tubes while purging with nitrogen gas, taking great care to minimize exposure to air, and quickly and tightly close the sample tube. (2) Submerge the tube in 37 °C water bath in a 37 °C room or incubator on the rotator. Incubate for 3 h or until solution has polymerized. (3) In a fume hood, extract the embedded sample from the gel (carefully take the sample out and remove extra gel pieces with gloved fingers). Hydrogel waste disposal should be conducted in accordance with all institutional, state and country regulations for hydrogel monomers and crosslinkers (for example, acrylamide and PFA). (4) Wash the sample with 50 ml clearing solution for 24 h at room temperature to dialyse out extra PFA, initiator and monomer. Wash the sample twice more with 50 ml for 24 h, each at 37 °C to further reduce residual PFA, initiator and monomer. Take care to dispose of this waste solution carefully as a biohazard.
ETC: (1) Construct the ETC chamber as described (http://CLARITYresourcecenter.org): place the sample in the chamber, and circulate the clearing solution through the chamber using the temperature-controlled water circulator, with 10–60V applied across the tissue (for example, brain) at 37–50 °C for several days to clear the sample. The clearing process is faster at higher voltage and temperature, but requires more power, limiting the number of clearing set-ups simultaneously operable by one power supply (typical power output maximum, 300 W). For example, four set-ups can be run simultaneously at 37 °C and 30 V, whereas only two set-ups can be run at 50 °C and 60 V, so circulator temperature and voltage should be chosen to meet practical considerations. In addition, too-high voltage operation could cause bubble formation in the tissue and deposit of black particles on the surface of tissue. Therefore, low voltage (10–40 V) is recommended. Note that lipids and biomolecules lacking functional groups required for conjugation, such as native phosphatidylinositol 4,5-bisphosphate or exogenous dextrans used for labelling, may be lost during this process. (2) After several days, wash the sample twice with of PBST (0.1% Triton X-100 in 1× PBS) twice for 24 h each.
Preparing the sample for imaging: (1) Incubate the sample in FocusClear or 80% glycerol solution for 2 days before imaging; these have refractive indices matching that of clarified tissue. Ensure there is sufficient solution surrounding the sample, and that evaporative losses do not occur. Protect the sample from light. (2) Take a clean glass slide and gently place it on a dust-free surface. (3) Take a small piece of BluTack putty and prepare constant-diameter worm shapes using gloved hands. Make the thickness uniform and about 1.5× the thickness of the sample (for example, if the sample is 1 mm, make the putty tube diameter 1.5 mm). (4) Place two tubes of putty horizontally across the vertical slide, leaving space in between for the tissue sample. Cut excess putty that protrudes off the slide. (5) Using a spatula, carefully take the sample and place it between the putty tubes in the middle of the slide. Pipette ~20 µl of FocusClear medium on top of the sample. (6) Carefully place a Willco dish (with the lipped side facing up) on top of the putty tubes. Press down on the glass part of the dish (keeping fingers over the putty to avoid glass shattering) carefully and slowly until contact is made with the sample and FocusClear medium. Ensure that there are no bubbles between the sample, medium, slide and dish. (7) Using a P200 pipette, carefully introduce more FocusClear to either side of the sealed chamber (from the liquid that surrounded the sample for incubation as it has been optically matched). Take great care not to introduce any bubbles. (8) Now that the whole chamber is filled with FocusClear, use PDMS sealant (Kwik-Sil) across both vertical openings between the putty, dish and slide to fully seal the chamber and prevent evaporation. (9) Place aluminium foil on top of the chamber and place it on a level surface (shielded from light to minimize photodamage). Leave the sample for 10–15 min to allow the PDMS sealant to polymerize fully. (10) Preparation is now ready for imaging.

The overall reaction scheme is shown in Figure 1, and is described in detail below both in a standard protocol for chemists, as well as a less technical protocol intended for biochemists without formal training in organic chemistry. Additional notes on the synthesis are provided as supplemental information. All of the procedures involving organic solvents should be performed in a fume hood, and some of the reagents are especially toxic (methyl iodide and dicyclohexylcarbodiimide). Proper safety equipment should be used to handle these compounds and care should be taken to minimize exposure. The procedures described below are identical for all four isotopic tags; although the molecular weights and densities of the methyl iodide vary, the differences are so small as to be negligible. The use of CH₃I results in the production of D0-TMAB-NHS (Figure 2), which was previously referred to as H9-TMAB-NHS22 (link). The use of methyl iodide with 3 deuterium atoms in place of the hydrogen atoms (Cd₃I) produces D9-TMAB-NHS, as previously described 22 (link);23 (link). The two new labels are produced from CH₂dI and CHd₂I and result in the formation of D3-TMAB-NHS and D6-TMAB-NHS, respectively (Figure 2).