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SWITCH (system-wide control of interaction time and kinetics of chemicals) is a simple, scalable, and generalizable tissue-processing method for proteomic imaging of intact biological systems. The SWITCH tissue-processing and clearing method provides access to high-dimensional multi-scale information that may help to understand health and disease from molecules to cells to entire systems.
Developed by the Chung Lab.
|32% Paraformaldehyde (PFA)||Electron Microscopy Sciences, 15714-S|
|50% Glutaraldehyde (GA)||Electron Microscopy Sciences, 16310|
|Potassium hydrogen phthalate||Sigma-Aldrich, P1088|
|Sodium azide||Sigma-Aldrich, S2002|
|Triton-X (TX) 100||Amresco, 0694|
|Sodium dodecyl sulfate (SDS)||Sigma-Aldrich, L3771|
|Sodium sulfite||Sigma-Aldrich, S0505|
|N- methyl-D-glucamine||Sigma-Aldrich, M2004|
|Diatrizoic acid||Sigma-Aldrich, D9268|
|60% iodixanol||Sigma-Aldrich, D1556|
This solution should be made fresh immediately prior to performing perfusion and kept on ice at all times. It is recommended to chill all of the separate ingredients before mixing the components.
|10X PBS||4 mL|
|32% PFA||5 mL|
|50% GA||0.8 mL|
Titrate a bottle of PBS to pH 3 using HCl. Create solutions of 0.1 M HCl in water and 0.1 M potassium hydrogen phthalate (KHP) in water. Finally, mix these solutions in a ratio of 2:1:1 (pH 3 PBS):(0.1 M HCl):(0.1 M KHP).
To this solution, add a stock solution of GA to make a final concentration of 4% GA. Ensure that this solution stays cold at all times. It is recommended to chill the solution before adding GA.
Add a stock solution of GA to PBS (pH 7.4) to make a final concentration of 1% GA. Ensure that this solution stays cold at all times. It is recommended to chill the PBS before adding GA.
To PBS, add TX-100 to a final concentration of 0.1% (v/v). Also, add sodium azide to a final concentration of 0.02% (w/v).
To PBS, add acetamide to a final concentration of 4% (w/v) and glycine to a final concentration of 4% (w/v).
Thermal clearing solution
To water, add SDS to a final concentration of 200 mM and sodium sulfite to a final concentration of 20 mM. This solution should be made fresh frequently, as the sulfites tend to degrade over time in solution.
To PBS, add SDS to a final concentration of 10 mM. Dissolve 1mg of DiD powder per 200 μL. This solution should be kept protected from light. Note: molecules similar to DiD can be used if other excitation/emission wavelengths are desired, so long as the molecule is sufficiently lipophilic.
To PBS, add SDS to a final concentration of 0.5 mM. This is most easily accomplished by diluting a stock solution of SDS. When adding large proportions of antibody to this solution (say, >1:10), care should be taken to account for the resulting change in SDS concentration.
Optical clearing solution
This solution consists of 23.5% (w/v) N-methyl-D-glucamine, 29.4% (w/v) diatrizoic acid, and 32.4% (w/v) iodixanol in water. Use a stir bar (or shake if necessary) to fully dissolve the powders at each step. Do not use heat when mixing the solution, as this will cause a color change.
This solution should be stored carefully to ensure that no water is lost, as just a small amount of evaporation will result in precipitation. Teflon tape can be used to increase the security of the bottle’s seal, and parafilm can be used around the cap.
It may be necessary to use a 60% iodixanol solution (see reagents list) rather than iodixanol powder, as it is not cheaply available. The optical clearing solution in the case would look as follows:
|47% Iodixanol solution in water*||10 mL|
|Diatrizoic acid||4.24 g|
Perfusion is the preferred method of tissue preservation. Using the perfusion technique of your choice
1b. SWITCH-mediated tissue preservation
If perfusion is not possible, the sample must be preserved using SWITCH.
Note: the timing for the fixation-OFF and -ON steps is dependent on the sample size and may need to be optimized from these starting values on a case-by-case basis. We found that these parameters worked well for banked human samples of roughly 0.5–1.0 cm thickness.
After fixation via either perfusion or SWITCH, the sample must be washed in PBST to remove unbound fixative molecules.
Note: if the sample needs to be cut, this should take place now before the sample is cleared.
Inactivated samples must next be incubated in thermal clearing solution to wash away remaining inactivation solution and to distribute sodium sulfite through the sample.
The length of time for incubation will depend on the size of the tissue. For mouse brain sections this is approximately two hours for a 100 µm section, up to several days for a whole mouse brain. It is important that the user regularly checks the progress in order to gauge the correct length of time for incubation. As a general rule, if the sample is placed over a grid or some letters/markings, you should be able to see the black lines through it when the sample is ready.
Other temperatures or methods of consistent heating may be used, but samples may deteriorate over time at higher temperatures.
Note: If a sample contains fluorophores that were genetically-encoded, introduced through viral injection, etc, then the sample may be cleared at 37 ˚C to preserve this fluorescence. The clearing process will take much longer at this low temperature, but temperatures higher than this will result in loss of fluorescence during clearing.
Note: Falcon tubes can become fragile over time in these conditions, so it is necessary to frequently check that the tubes have not begun to leak.
4a. SWITCH-mediated myelinated fiber-labelling
After a sample has been cleared, SWITCH-mediated labelling is possible. Myelinated fibers can be readily visualized with the lipophilic DiD fluorescent molecule.
We have also observed that tomato lectin and nuclear stains such as DAPI or Syto16 can be used with this SWITCH approach.
4b. SWITCH-mediated immunolabelling
After a sample has been cleared, SWITCH-mediated labeling is possible.
After labeling, the sample must be equilibrated in a refractive index-matching solution to facilitate imaging.
After imaging, the optical clearing solution should be washed out of the sample with thermal clearing solution.
Murray, E., Hun, J., Goodwin, D., Ku, T. & Swaney, J. Simple, scalable proteomic imaging for high-dimensional profiling of intact systems. Cell 163, 1500–1514 (2015).