Clearing Protocol

These are the materials that you will need:

  
	Name
    Vendor
    Catalogue Number
  

    Boric acid
    Sigma
    B7901
  

    Lithium Hydroxide
    Sigma
    254274
  

    Sodium Dodecyl Sulfate
    Sigma
    L3771
  

    Spectra/Por 1
    Spectrum Labs
    132655
  

    Custom constructed device
    N/A
    N/A
  

Granted, the hardest part will be constructing your device. We've included a blueprint for the device in the supplementary materials of the paper. We're also working with a manufacturer to produce these devices, so stay tuned.

You'd need to make the following solutions:

Inner: 200 mM SDS, 10 mM Lithium Hydroxide, 20 mM Boric Acid
Outer: 10 mM SDS, 10 mM Lithium Hydroxide, 20 mM Boric Acid

There are two different solutions: Inner and Outer. The Inner solution has the optimum concentration of SDS necessary for rapid tissue clearing. The Outer solution has just enough SDS to maintain micellar equilibrium with the Inner solution SDS. Having these two solutions significantly decreases SDS degradation - meaning less maintenance and lower costs.

Once everything is ready to go, pour the Inner solution into the Inner reservoir and the Outer solution into the Outer reservoir. Put the brain inside of the rotating "sample chamber" (which is just a cylindrical nylon mesh to make sure the tissue doesn't float away). Then, apply the maximum voltage you can across the electrodes that does not cause (1) bubble formation in the tissue, (2) runaway Joule heating (i.e. temperate keeps rising and rising), or (3) damage to the device (the membranes, plastic parts, or the electrodes). There isn't a magical voltage for this - it's all trial and error. It depends on your device geometry (namely, distance between the electrodes) and your tissue. This is one of the reasons why we're trying to produce standardized devices with well-characterized properties and easy-to-follow protocols.