From CLARITY Wiki
After hydrogel tissue embedding, the tissue sample is placed in clearing solution to begin the process of lipid clearing. At this point, the tissue biomolecules, other than lipids, are chemically attached to a hydrogel matrix within the tissue. The lipids are the main source of light scattering within the tissue sample, causing it to be opaque. The clearing solution contains an ionic detergent that collects the lipids and transports them out of the tissue hydrogel, eventually leading to a clear and transparent tissue sample that still contains fine structural details from the biomolecules that are crosslinked in place.
Clearing solution wash
After hydrogel tissue embedding, all tissue samples undergo a clearing solution wash step for a minimum of 2 days. The purpose of the washing step is to dialyze out the excess hydrogel monomers (formaldehyde, acrylamide, bis-acrylamide, thermal initiator) from inside the tissue. Removal of the monomers is necessary to prevent further polymerization and crosslinking inside the tissue. Also, as a couple of the monomers are toxic, it is best to first rinse them out and dispose of them properly as it will greatly ease handling of the sample and clearing solution during ETC.
After removing the newly embedded tissue sample from the bulk hydrogel, place the sample in a container with 50 mL of clearing solution. Incubate at room temperature or 37°C overnight on a shaker/rotator plate.
Replace the 50 mL of clearing solution after 1 day and continue incubation at 37°C with shaking.
Following each 50 mL wash, discard the clearing solution in a liquid waste container in a fume hood. Do not pour the clearing solution from the first 2 washes down the sink as it contains toxic PFA and acrylamide monomers.
- Note: If planing to section or slice the tissue sample, it is best to complete this immediately after hydrogel embedding before washing with the clearing solution.
Clearing methods and strategies
After the initial 2-day wash in clearing solution to rinse out the hydrogel monomers, the sample is kept in the clearing solution in order to remove the lipids and unattached biomolecules from the hydrogel-embedded tissue. There are two methods for clearing: passive clearing and electrophoretic tissue clearing (ETC). One or both of these methods can be applied to clear the tissue sample. In certain cases, one method may be preferred over another, as discussed in the clearing strategies section below.
The clearing solution, which contains an ionic detergent that forms micelles in solution, is used to clarify the hydrogel-embedded tissue by removing the unattached fatty lipids. Sample clearing can be performed in two ways:
- Passive clearing - Basically a continuation of the clearing solution wash step, this method relies on passive diffusion of the lipid-collecting micelles in and out of the tissue by incubating the sample in clearing solution until it appears visually clear.
- Electrophoretic tissue clearing (ETC) - Taking advantage of the ionic charge on the SDS micelles in the clearing solution, ETC uses an electric field around the sample to actively diffuse the micelles in and out of the tissue for faster clearing.
The best clearing method to use for a given tissue sample can depend on an array of variables including sample size, labelling method, processing time constraints, desired imaging depth, and the overall purpose of the tissue sample (i.e. sample used for pilot testing or screening vs. sample for specific data collection).
100% passive clearing
Passive clearing is the simplest and cheapest method of clearing, and it is also the gentlest on the sample tissue. Empirically, samples that undergo only passive clearing have provided the greatest imaging depths and best immunostaining results. In addition, passively cleared samples achieve the best overall transparency. The drawback of using only passive clearing, though, is that it is usually a multi-week long process, the slowest of the clearing strategies.
A 100% passive clearing strategy is recommended for:
- Small and/or fragile samples (may be risky to place in ETC chamber under high flow)
- Samples that require immunostaining
- Samples that require large imaging depths (for example, dorsal to ventral of a whole mouse brain, ~6 mm)
- High-quality data collection samples
It is also recommended when setting up CLARITY to use passive clearing on a couple initial tissue samples. This will help with troubleshooting the CLARITY process. If encountering difficulties clearing the sample using ETC, samples undergoing passive clearing can be used as a reference to determine if there is a problem earlier in the process during hydrogel embedding (passive clearing samples are not clearing either) or if the problem is with the ETC set-up and conditions.
ETC is the fastest method of tissue clearing. However, it involves exposing the tissue sample to a high flow rate and an electric field, so there is a risk of sample damage. The start-up equipment cost is high, but if well-maintained, ETC equipment will not need to be replaced very often. It is difficult to obtain complete sample transparency using only ETC as it requires longer exposure times, which can lead to significant yellowing of the sample tissue.
An ETC clearing strategy is recommended for:
- Larger/whole tissue samples (may take months to clear passively)
- Transgenic or pre-labelled samples (markers are attached to the hydrogel during embedding)
- Samples that need to be processed quickly
Passive clearing/ETC combination
A happy medium to receive the benefits of both passive clearing (gentler on sample, better transparency) and ETC (time-saving) is to combine the two methods. ETC helps to clear out the majority of the lipids quickly, and passive clearing takes care of the rest. There is no right or wrong way to combine passive and ETC clearing, but the following strategy works well:
- Passive clearing (5-10 days) - Starting with passive clearing helps to gently open up and swell the tissue sample so that it is more responsive to ETC. The outer edge of the tissue will start to look visually clear during this step.
- ETC (1-3 days) - A shorter period of ETC will help to remove a majority of lipids from the tissue sample without causing a significant amount of sample yellowing. There is still a small amount of risk associated with leaving the sample in the ETC set-up, but with proper ETC conditions, this risk is minimal.
- Passive clearing (1-2 weeks, or until sample appears clear) - If the sample does not appear clear upon removal from the ETC chamber, passive clearing afterwards helps to remove the residual lipids from the tissue. Visually check for sample clarity by holding the sample up to the light to test for transparency. Once one is able to see through the entire span of tissue, the sample can be removed from the clearing solution and placed in PBST for the next CLARITY step.
- Note: Passive clearing removes lipids from the outside edge of the tissue first, such that the inner core of the sample is the last to appear transparent. In comparison, ETC clears the whole tissue sample at the same time because it actively pushes the clearing solution from one side of the tissue through to the other side. For this reason, a tissue cleared using ETC may still look opaque upon removal from the chamber (when there are still residual lipids throughout the entire sample), but then turn completely see-through all at once after a few days of passive clearing to remove the leftover lipids.
- Note: The time recommendations work well for a mouse brain tissue sample but may need more adjustment for alternative tissues.
A passive clearing/ETC combination strategy is recommended for:
- Larger/whole tissue samples
- Smaller samples that need fast processing
- Transgenic or pre-labeled samples
- Samples that require average imaging depths (0-3 mm)
PBST buffer wash after clearing
After sample clearing, all tissue samples undergo a PBST (0.1% TritonX in 1X PBS) buffer wash step (similar to the clearing solution wash step) for a minimum of 2 days. The purpose of the washing step is to rinse out the SDS micelles from inside the hydrogel-embedded tissue sample. The presence of micelles inside the tissue could interfere with antibody binding during immunostaining or with the mounting solution during imaging (causing irreversible precipitation), so it is best to wash the residual clearing solution out of the tissue sample before proceeding with those steps.
When the tissue sample appears visually see-through (sample will be swollen to a larger size and not necessarily fully transparent), remove it from the ETC chamber or clearing solution and place it in a container with 50 mL of PBST (0.1% TritonX in PBS). Incubate at room temperature or 37°:C overnight on a shaker/rotator plate.
Replace the 50 mL of PBST after 1 day and continue incubation with shaking. Repeat as desired or store the sample until ready for immunostaining or imaging.
- Note: Samples swollen in PBST will remain swollen and possibly turn cloudier than they appeared in clearing solution. This is due to refractive index differences between the tissue and PBS.
- Note: TritonX is an important component in the buffer as it is a detergent, similar to SDS, and therefore more efficient in removal of SDS micelles than PBS buffer alone. Do not substitute PBS for PBST when transferring the sample from clearing solution.
Hydrogel-embedded tissue samples can be stored in the clearing solution for several months (if needed) as they undergo passive clearing. Passive clearing will go quicker at higher temperatures (37°C, for example), but room temperature or lower is safer for long-term storage (more than a few months).
Once samples reach near-complete visual transparency, it is recommended to store them at room temperature or lower or to transfer the sample to PBST for storage. Sample storage at 37°C for many months in clearing solution has been observed to have negative effects on the hydrogel integrity and maintained tissue structural details.
CLARITY samples can be stored indefinitely in PBST or PBS buffer. Store samples at room temperature or 4°C. A small amount of sodium azide can be added to the solution for long-term storage to prevent bacterial growth.