CLARITY

This is the first installment in "Don't Sweat the Technique(s)," a series dedicated to explaining and reviewing neuroscience research techniques without much technical jargon. I was inspired to start this series, in part, due to the awe and confusion I felt while learning CLARITY; now, I use the technique regularly and help others learn it themselves!

Within each installment of this series, I'll break down techniques into useful background, and a translation of the published protocol.

Background

So, what is CLARITY? Well, the acronym itself stands for "Clear Lipid-exchanged Anatomically Rigid Imaging/immunostaining-compatible Tissue hYdrogel." But I didn't find that very helpful when initially learning about the technique. In simpler terms, it is a method used to immunolabel and image brains (or other tissues), so that you can actually keep the tissue intact instead of sectioning it (this is especially helpful when you are studying circuits, like me!).

The usefulness of this technique rests on the ability to form a gel out of the proteins and nucleic acids within the brain, along with acrylamide monomers you introduce into the tissue when you perform a transcardial perfusion of the animal, leaving the lipids to be washed away. If you can't form a gel matrix within the brain, you cannot successfully wash out its lipids, and imaging it will be difficult.

Protocol

The first step in the CLARITY protocol actually happens before you even perfuse your animal: you must prepare your monomer solution. Monomer/hydrogel solution replaces the paraformaldehyde (PFA) solution you typically use to fix tissues, but really isn't all that different from it. Monomer solution consists of...

• 4% Acrylamide
• 0.05% VA-044 Initiator
• 4% Paraformaldehyde
• 1X PBS

Assuming you've perfused tissue with PFA before, you already know that paraformaldehyde fixes tissues and the PBS acts as a buffer, but you'll note the presence of acrylamide and VA-044 in the monomer solution. The acrylamide monomers will link up with the formaldehyde in solution, which will then link to amine-containing molecules within the tissue (proteins and nucleic acids), and the VA-044 will initiate gel formation when under the proper conditions.

After preparing monomer solution, make 40 mL aliquots of it and store at -20C or leave on ice for immediate use. For one mouse brain, you need 20 mL of ice cold monomer solution for perfusion and 20 mL of cold monomer solution for overnight incubation of the brain.