From CLARITY Wiki
Staining with either antibodies or dyes is performed following sample clearing and a two day PBST buffer wash. Afterwards, sample imaging using three-dimensional techniques, such as confocal microscopy, can be performed. Tissue samples can then be stored in PBST or processed for additional rounds of immunostaining and imaging.
Guidelines for immunostaining
Successful immunostaining results are often strongly dependent on the antibody choice. Protocol modifications and optimizations may need to be performed for different antibodies, and so one standard procedure can not be applied. The following are guidelines for obtaining successful staining results.
- Use long enough incubation times - Multiple days for incubation will likely be needed. For a 1 mm section, a good starting point is: 2 day incubation in primary antibody, 1 day wash with buffer, 2 day incubation in secondary antibody, 1 day wash with buffer.
- Use high antibody concentrations - Start with antibody concentrations in the range of 10 μg/mL (1:100) to 20 μg/mL (1:50), and lower as needed.
- Use high temperature - Incubating at 37°C will promote quicker antibody penetration into the clarified tissue sample. If antibody binding is a problem, though, incubation at lower temperatures (4°C, for example) can be done for longer times. A combination of temperatures (37°C followed by 4°C) may combine the benefits of both.
- PBST buffer - While the sample can be transferred to other buffers for immunostaining, PBST buffer is known to work well.
- Passive clearing - Immunostaining seems to work best in samples that have been passively cleared as opposed to ETC. It is recommended to use slightly longer passive clearing incubation times to get the sample to near transparency even in the clearing solution.
- Smaller sections - 1-2 mm thick sections will provide the best results. Whole tissue staining will need signficantly longer incubation times and antibodies may still not be able to fully penetrate to the core of the sample (a whole mouse brain, for example, took 2 weeks incubation in each antibody with 1 week long buffer wash steps).
- Blocking agent - Use of a blocking agent in brain tissue has not shown an obvious difference in immunostaining results and is probably not needed. This may not be the case for other tissue types.
- Hydrogel solution modification - The hydrogel solution can be modified to reduce the amount of crosslinking in the hydrogel-embedded tissue and therefore allow faster and better penetration of antibodies during staining.
Recommended antibodies and dyes
The following is a list of antibodies and dyes that should provide reliable staining results. These recommendations may be useful to start with when testing out the CLARITY method for the first time.
- Parvalbumin (abcam)
- Tyrosine Hydroxylase (abcam)
- Propidium Iodide
- DAPI (may wash out in mounting solution after 1-2 days)
- Neurofilament NF-H (chicken antibody from Aves)
The stable framework of the clarified tissue, whereby the innate biomolecules are chemically attached to the hydrogel network, enables multiple rounds of molecular phenotyping without damage to the preserved structure. In fact, the clearing solution containing SDS micelles that was originally used for tissue lipid clearing can be used following the first round of immunostaining to denature antibodies, disrupt binding, and wash the first round of antibodies and molecular labels out of the hydrogel-embedded tissue.
- Remove sample from imaging chamber, place in 50 mL PBST and incubate at room temperature or 37°C overnight with shaking
- Place sample in 50 mL of clearing solution and incubate at 60°C overnight with shaking to wash out first round of antibodies
- Place sample in 50 mL PBST and incubate at room temperature or 37°C overnight with shaking to wash out clearing solution
- Complete next round of immunostaining and imaging