Hydrogel Embedding

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(Procedure and completion point)
(Procedure and completion point)
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*''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:'' 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 embedding. However, this will not occur if certain changes to the hydrogel solution, namely removal of bis-acrylamide or reduction of acrylamide concentration, are made.
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*''Note:'' Gelation of the solution surrounding the tissue sample is an excellent indicator of successful embedding. However, this will not occur if certain [[Solutions#Changes to the hydrogel solution composition|changes to the hydrogel solution]], namely removal of bis-acrylamide or reduction of acrylamide concentration, are made.
   
 
====Importance of 3 hour incubation====
 
====Importance of 3 hour incubation====

Revision as of 23:35, 11 April 2014

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.

Contents

Degassing

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.

Procedure

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Degassing chamber and vacuum pump

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).

  1. Place the conical tube containing the tissue sample in hydrogel solution on a rack in the desiccation chamber.
  2. 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.
  3. 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.)
  4. 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.
  5. 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.
  6. 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.
  7. 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.

Alternatives

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

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Hydrogel polymerization schematic

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 embedding. However, this will not occur if certain changes to the hydrogel solution, namely removal of bis-acrylamide or reduction of acrylamide concentration, are made.

Importance of 3 hour incubation

-3 hours (or a little over) without oxygen present is sufficient enough time to fully polymerize and crosslink hydrogel monomers into a gel that can be handled but is still soft and porous - the polymerization reaction does not go to completion in 3 hours and will continue if left at high temperature for longer - if left for longer times, gel may become too stiff - not only will gel removal be much more difficult, but a stiffer gel will be much more crosslinked and significantly less porous, meaning much slower clearing and staining processes if they are even able to clear at all

Troubleshooting

If a hydrogel is formed after 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.

  • 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 and temperatures lower than 37°C, even 34-35°C, can prevent polymerization

Suggestions

Excess gel removal

Purpose of gel removal

Procedure

Sample slicing/sectioning