From CLARITY Wiki
After the tissue samples have been prepared via perfusion and/or hydrogel solution incubation, the next step involves polymerizing the hydrogel monomers to embed the tissue in a hydrogel matrix. The procedure for hydrogel tissue embedding starts with a degassing step to thwart oxygen inhibition followed by sample incubation at high temperature to polymerize the hydrogel monomers. Afterwards, excess gel is removed from the sample surface. Immediately following hydrogel embedding is the best time in the CLARITY process to complete sample slicing or sectioning if desired. The purpose, procedure, and troubleshooting suggestions for each of the hydrogel embedding steps are discussed below.
Purpose of degassing
Hydrogel network formation is instigated by a thermal initiator present in the hydrogel solution. At low temperature (4°C), the initiator molecule is inert, which helps provide ample time for hydrogel monomers to distribute uniformly throughout the tissue during incubation. At higher temperatures, the initiator molecule becomes more active and creates free radicals in the hydrogel solution which initiates free radical polymerization of the acrylamide monomers. Oxygen acts as a free radical quencher, meaning that acrylamide polymerization is inhibited in the presence of oxygen. So, for efficient hydrogel polymerization, oxygen needs to be removed from the container beforehand.
It should be noted that acrylamide polymerization can occur slowly even in the presence of oxygen. This will happen, for instance, if the hydrogel solution is left out at room temperature (or even in the refrigerator after a few weeks!). To quickly get uniform and consistently polymerized hydrogels, though, oxygen removal is necessary.
The following degassing procedure uses a desiccation chamber (in a fume hood) hooked up to a nitrogen tank as well as a vacuum pump. A vacuum is applied to the chamber to remove all oxygen and replace it with pure nitrogen gas. A 50 mL conical tube is described as the sample container, but any container with a wide-mouth (i.e. not necked) that can be sealed shut will work (generally, use the same container that the sample sits in during hydrogel solution incubation).
- Place the conical tube containing the tissue sample in hydrogel solution on a rack in the desiccation chamber.
- Twist off the cap, but leave it on top of the conical tube such that the container is sufficiently open enough to allow gas exchange. Place the lid on to the desiccation chamber.
- Adjust the desiccation chamber control valve to allow flow from the nitrogen tank to the desiccation chamber, and turn on the nitrogen tank. (A brief period before switching to vacuum is fine; the purpose here is to fill the inlet tubing to the nitrogen tank with gas to avoid introducing air into the chamber after the vacuum step.)
- Turn on the vacuum pump. Slowly turn the control valve to switch to flow between only the vacuum pump and the desiccation chamber. Verify that the chamber is under full vacuum by gently tugging on the chamber lid. Keep the vacuum on for 10 minutes.
- After 10 minutes, turn the vacuum pump off. Slowly turn the control valve to allow nitrogen gas to flow into the desiccation chamber. Let nitrogen flow in for several seconds to fill the chamber with gas.
- Carefully lift the desiccation chamber lid just enough to reach the tube while continuing to purge with nitrogen gas. Quickly twist the conical tube cap back on while taking great care to minimize exposure to air.
- Remove the conical tube from the desiccation chamber and check to make sure it is sealed tight. Turn off the nitrogen gas tank.
- Note: If the sample tube cap gets knocked off when reaching to close it after applying vacuum, it is probably best to repeat the entire degassing step before moving on to high temperature incubation. The increased air exposure to the sample container while picking up the cap and placing it back on could lead to observable oxygen inhibition (i.e. the gel will not be fully polymerized after 3 hours of incubation). If ever concerned that the sample container was exposed to more than a minimum amount of air after degassing, it is best to repeat the degassing rather than risk inhibiting polymerization (and thus having to repeat both steps again later).
- Note: It is best to perform degassing on one to two samples at a time. This ensures minimum exposure of the sample containers to air when tightening the caps during the nitrogen purge.
- Note: Any inert gas can be used instead of nitrogen.
There are several modifications or alternatives to the degassing procedure that can also be used to remove oxygen inhibition during hydrogel polymerization. Some of these may be easier to use based on available lab equipment and materials. Basically, if a non-flowing hydrogel is obtained after about 3 hours of incubation at 37°C (i.e. a gel should form in the solution surrounding the tissue as long as bisacrylamide is present), the degassing procedure used can be considered successful.
- Hook up to house vacuum - Instead of using a vacuum pump, the desiccation chamber can be hooked up to the house vacuum system. For safety, it is recommended to use a solvent trap in case hydrogel solution accidentally gets sucked into the vacuum line.
- Purge with inert gas (no vacuum) - Rather than pulling a vacuum to remove oxygen and then replace it with inert gas, inert gas can be purged into the system directly while air escapes.
- Transfer to smaller container with no air - Air can be "removed" by transferring the sample and hydrogel solution to a smaller container such that the volume of solution completely fills the container and there is little to no excess air inside.
- Pour oil on top of solution - A layer of oil (peanut or mineral oil, for example) can be poured on top of the hydrogel solution to prevent oxygen diffusion into the solution during polymerization. The oil layer will sit on top of the solution and can be easily poured off from the solid gel after polymerization is complete.
High temperature incubation
Purpose of high temperature incubation
The thermal initiator in the hydrogel solution, which starts polymerization once it becomes active, is highly temperature dependent. It is inert and non-reactive at low temperature and becomes increasingly more reactive as the temperature is increased. This is ideal for performing CLARITY as the tissue can be kept at low temperature (4°C) for a few days to allow time for the hydrogel monomers to diffuse in. Once the monomers are uniformly distributed and ready for polymerization, high temperature incubation (37°C) activates the thermal initiator and triggers free radical polymerization of the acrylamide monomers. Without oxygen present as an inhibitor, acrylamide polymerization occurs quite quickly at 37°C, and a complete hydrogel is formed in about 3 hours. Biomolecules such as proteins, DNA, and RNA are linked to the acrylamide monomers via formaldehyde, so acrylamide polymerization forms a hydrogel-tissue hybrid network with the biomolecules covalently attached to the polyacrylamide matrix in their native positions.
Procedure and completion point
After degassing, submerge the tissue sample container in a temperature-controlled 37°C water bath. It is recommended that the water bath be placed on a rotator/shaker plate in either a warm room or incubator; however, shaking or stirring is not necessary. A water bath is highly recommended because water has a higher rate of heat transfer compared to air, so it will more efficiently heat up the hydrogel solution container for quick gelation.
Incubate the sample at 37°C for about 3 hours. The polymerization is complete when the solution in the container no longer flows. In some instances, a very small amount of liquid may remain at the top of the hydrogel (at the air/solution interface) due to a minor amount of oxygen inhibition.
- Note: The 3 hour incubation time for polymerization does not change based on the size of the tissue sample or different sample preparation conditions.
- Note: Gelation of the solution surrounding the tissue sample is an excellent indicator of successful tissue embedding. However, this will not occur if certain changes to the hydrogel solution are made, namely removal of bis-acrylamide or reduction of acrylamide/bis-acrylamide concentration. In these cases, there will not be enough crosslinkers in the solution surrounding the tissue to form a gel, but a hydrogel matrix will still form inside the tissue due to all the linked biomolecules present to hold it together. Though the surrounding hydrogel solution will not gel during incubation, it should get more viscous due to the polymerized acrylamide.
Importance of 3 hour incubation
3 hours (or a little more) without oxygen present is sufficient enough time to fully polymerize and crosslink the hydrogel monomers into a gel that is rigid enough to be handled, but still soft and porous. It is important to note that the polymerization reaction does not go to completion in 3 hours and will continue if the sample container is left at high temperature for longer. While an extra hour or two at high temperature may not make a significant difference, if the sample is left for much longer times, overnight for instance, the resulting hydrogel may become too stiff for CLARITY processing. Not only will excess gel removal from the tissue be more difficult, but a stiffer gel will be much more crosslinked and significantly less porous, causing the tissue clearing and staining steps which rely on molecular diffusion through the sample to be much slower, if the tissue sample is even able to clear at all. Thus, it is not recommended to leave samples under high temperature incubation for time periods significantly longer than 3 hours.
If a hydrogel is formed after high temperature incubation for 3 hours, the degassing and incubation steps worked as designed. However, if little to no gel is present after 3 hours and the solution is still very much a solution, there could be a couple reasons that polymerization is not working (assuming, of course, that the bis-acrylamide concentration in the hydrogel solution was not reduced to begin with, as discussed above).
- Oxygen inhibition - Too much air seeped into the container before the cap was sealed after degassing or degassing was not done for a long enough time
- Temperature too low - Incubation at 37°C in a water bath is critical for fast polymerization; temperatures lower than 37°C, even 34-35°C, can prevent polymerization within a 3 hour time frame
If the hydrogel solution has not formed a gel after 3 hours, check the water bath/incubator temperature to make sure it has not fallen from 37°C. Repeat the degassing procedure (or use an alternative one) and repeat the high temperature incubation for another 3 hours. The sample can be returned to the refrigerator overnight before repeating the embedding steps if necessary.
Excess gel removal
During high temperature incubation, the hydrogel monomers inside and outside the tissue polymerize and crosslink to form a hydrogel matrix. A large excess of hydrogel will be formed in the solution surrounding the tissue, and the last step of embedding is to remove this excess gel. As noted in the high temperature incubation procedure above, this step is not necessary if bis-acrylamide is left out of the hydrogel solution or if its concentration is significantly reduced, in which case the tissue sample can be easily scooped out of the solution following 37°C incubation.
Purpose of gel removal
The presence of bis-acrylamide (a small chemical crosslinker) in the hydrogel solution causes the solution surrounding the tissue sample to also form a hydrogel during polymerization at 37°C. There are two main reasons that it is important to remove as much of this excess gel from the surface of the tissue as possible. The first reason is that an extra layer of gel around the tissue will hinder the remaining CLARITY processing steps. Clearing and immunostaining times will increase since the excess gel will act as an additional barrier for diffusion of molecules in and out of the tissue sample. Secondly, it has been observed that extra gel on the tissue surface can sometimes turn black during ETC, after which it will no longer be separable from the cleared tissue sample that will have a similar hydrogel consistency.
In a fume hood, run a spatula along the inside of the sample container to disconnect the bulk gel sample and gently guide it out of the container. Use gloved fingers to carefully separate the sample from the excess bulk hydrogel. Gently rub the tissue surface with dry gloved fingers or tissue (e.g. Kimwipes) to remove the extra gel pieces. When no more gel lifts off the surface of the tissue, place the sample in a container with 50 mL of clearing solution to begin washing out the excess hydrogel monomers from inside the tissue.
- Note: Hydrogel waste disposal should be conducted in accordance with all institutional, state, and country regulations for the hydrogel monomers and crosslinkers (acrylamide and PFA). Place the excess hydrogel pieces as well as any materials that contact the hydrogel (conical tube, gloves, tissue, etc) into a solid waste container.
The best time to section the tissue sample when processing with CLARITY is immediately following hydrogel tissue embedding, before placing the sample in the clearing solution. At this point, the tissue sample still contains all its lipid membranes in addition to a tissue-crosslinked hydrogel matrix, so the tissue is particularly robust and easy to handle. Also, the sample is still its anatomical size. After lipid clearing, samples in either clearing solution or PBST buffer will be swollen to a larger size. Removal of the lipids will also make the tissue much softer and more difficult to handle, especially for precise and reliable sectioning.
Samples can be sectioned manually, using a vibratome, or using a tissue matrix with razor blades. After sectioning, place the tissue samples in 50 mL of clearing solution (no change in procedure is needed from whole tissue samples).
Slicing or sectioning is highly recommended if only studying smaller subsections of a tissue sample. Smaller tissue sections will clear much faster than whole tissues, and they may clear relatively quickly passively, without even employing ETC. Immunostaining is also much easier and faster for thin tissue sections (<2 mm thick).