Labeling Protocol

These are the materials that you will need:

  
	Name
    Vendor
    Catalogue Number
  

    Boric acid
    Sigma
    B7901
  

    Lithium Hydroxide
    Sigma
    254274
  

    Molecular Probes
    Any
    Any
  

    Spectra/Por 1
    Spectrum Labs
    132655
  

    Custom constructed device
    N/A
    N/A
  

Again, 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:

Electrophoresis: 50 mM Lithium Hydroxide, 25 mM Boric Acid
Staining: 50 mM Lithium Hydroxide, 25 mM Boric Acid, 1% Triton-X 100, 0.02% Sodium Azide

Staining solution is the same as Electrophoresis solution, except that it contains 1% Triton-X 100 to minimize the adsorption of molecular probes onto the membranes and to permeabilize the tissue as well as sodium azide to prevent bacterial growth.

Before you begin labeling, you need to make sure the tissue contains no SDS, which interferes with labeling. To do that, we recommend you wash your tissue three times over three days in the Staining solution.

Once you have everything, pour the Electrophoresis solution into the Electrophoresis reservoir. Then, add enough Staining solution into the sample chamber to completely immerse the tissue. Pipet mix molecular probes into the Staining solution. 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). This is similar to clearing above. After about 24 hours (to make sure the labeling is done), take out the tissue and incubate it in your desired optical clearing solution. We use what we call PROTOS. You can find details about PROTOS in an upcoming post.