Sample Clearing

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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.

Contents

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.

Clearing methods and strategies

Methods

-passive clearing (continuation of clearing solution washing) -ETC

Clearing strategies

-dependent on sample size, labeling method, time, desired image depth and resolution, purpose of tissue sample -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


PBST buffer wash after clearing

Sample storage