From CLARITY Wiki
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.
Start by reviewing the CLARITY timeline which details the different steps involved in clarifying a tissue sample. Take note that while many of the steps require multiple days, most of that time is spent letting the sample incubate in a solution. Several of the steps can be modified, if not removed altogether, following the suggestions below.
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.
- 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. While the method does take longer, using smaller tissue sample sections to begin with will significantly reduce the clearing time.
- Incubate at higher temperatures (35-55°C) for passive clearing. Higher temperature incubation with gentle shaking will help speed up the clearing process. Routinely replacing the clearing solution will also help. Visually check the sample for clearing by holding it up to the light to look for complete transparency across the tissue.
Staining and Fluorescent Labelling
- If possible, start with transgenic tissue samples containing fluorescent proteins as the proteins will be incorporated into the hydrogel network and no additional staining will be needed for imaging. The best samples to try first should have high fluorescent expression levels and a strong signal.
- If staining is needed for visualization, start with 1 mm (or thinner) sections. Smaller samples provide the fastest and easiest penetration of antibodies and dyes.
- Start with antibodies and dyes that are familiar and expected to have good binding and a strong signal. This will help in developing a reliable staining procedure and verifying its success in clarified tissue before moving on to more specified stains.
- Again, use passive clearing as it has empirically shown to be the best method for good immunostaining results.
- Do not attempt to image the clarified sample using regular light microscopy. Since the sample is visually transparent, fluorescence will be emitted from every focal plane of the sample, and thus it will appear as a fluorescent blob that could be mistaken for autofluorescence. Use confocal microscopy to look for signal and image the sample.
- If hesitant to purchase FocusClear as a mounting solution because of the price, mount the sample in glycerol instead. Glycerol is not an ideal mounting solution for thick or whole tissue samples, but for small sections (<1 mm thick) it should work fine.