CLARITY may seem like an intimidating technique requiring a lot of time, effort, and expensive equipment to get it started and working consistently. However, many of the CLARITY steps can be simplified to make the process easier to get started.

The following recommendations are useful for getting started with CLARITY and getting a feel for performing each step and what to expect from the tissue sample in each stage. Once confident that the technique is working as it should, modifications that may increase complexity can be added. Depending on the application, the simplified CLARITY approach may be all that is needed.

Hydrogel Tissue Embedding

• Use the standard hydrogel solution containing bis-acrylamide. Modifying the hydrogel solution may be useful for certain types of samples, but it is best to start with the standard solution when attempting hydrogel embedding for the first couple times. The standard solution should form a tangible bulk hydrogel around the tissue sample after about three hours of high temperature incubation, which will indicate that the hydrogel embedding are working properly.

• If degassing supplies, such as nitrogen gas, are not readily available or the procedure seems complicated and inconsistent, try an alternative method to prevent oxygen inhibition during polymerization. Transferring the solution to a smaller container with no air or adding oil to the top of the solution may be simpler methods that work as efficiently as degassing.

• Be sure not to leave sample for much longer than about three hours during high temperature incubation or the gel may over-polymerize and the sample will be difficult to clear.

Sample Clearing

• Use smaller samples by sectioning the tissue samples immediately after hydrogel embedding. 1-2 mm thick samples will clear much faster and be much easier to use during sample staining and/or imaging.

• Start with passive clearing. Of the two clearing methods passive clearing is the easiest to do (simply sample incubation in clearing solution) and does not require any extra equipment like ETC.