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.

Procedure

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.

Methods

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.

Clearing strategies

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 resolution, and the overall purpose of the tissue sample (i.e. sample used for pilot testing or screening vs. sample for specific data collection). Recommendations for when to apply passive clearing or ETC depending on the tissue sample are listed below.

Passive clearing/ETC combination

-100% passive clearing - recommended for immunostaining, small and/or fragile samples, long-range imaging - need more time to process -ETC - recommended for whole/larger tissue samples, tissues that need to be processed quickly -passive clearing/ETC combo