It stands for Stabilization to Harsh conditions via Intramolecular Epoxide Linkages to prevent Degradation.
SHIELD protects a tissue sample and proteins inside the sample from environmental stress (heat and chemicals) without causing autofluorescence. The polyepoxy molecules crosslink surface-exposed protein residues with multiple flexible intramolecular bonds to provide structural reinforcement to enhance the stability of proteins.
You can find the SHIELD protocol here, beginning on page 3. In short, SHIELD uses a SWITCH-like protocol, where the epoxy is distributed throughout the sample evenly with an OFF Solution, then reacted with the tissue in an ON Solution. If you can perfuse, perfuse the animal with the perfusion solution (SHIELD Epoxy + SHIELD Buffer + PFA + DI water) and incubate in that solution. Then incubate in SHIELD-OFF (Epoxy + Buffer + DI water). Finally, incubate in SHIELD-ON. If you can’t perfuse, incubate the sample in SHIELD-OFF (Epoxy + Buffer + DI water), then incubate in SHIELD-ON.
No, you don’t need to perfuse, although perfusion produces the best preservation results. You can post-fix samples if they are already fixed with PFA.
No, there is no degassing step required.
SHIELD tissue has low autofluorescence and facilitates high signal-to-noise ratio of fluorescent protein-labeled tissues. Autofluorescence in SHIELD is much lower than in SWITCH and DISCO-treated tissues.
Any tissues that can be preserved with CLARITY can also be preserved with SHIELD. This includes brain, liver, lung, heart, kidney, pancreas, skin, etc.
SHIELD has been most extensively tested with mouse and rat tissue, and is also compatible with marmoset and human tissues as well as cultured tissues like organoids.
Yes, SHIELD preserves mRNAs for detection using fluorescence in situ hybridization (FISH).
The kit is priced at $350 for a 250 mL set and $550 for a 500 mL set. This comes out to ~$35/mouse. Discounts are available for bulk purchases, and you may sign up for a free trial to try it out.
A 250-mL kit allows for 8 mice perfusions or 1 rat perfusion. The 500-mL kit allows for 16 mice perfusions or 2 rat perfusions. However, if you are post-fixing tissues, the volume of solution used is smaller so you can preserve more samples (perfusion step not needed).
No, SHIELD does not require any specialized equipment, although an incubator or water bath that can maintain 50-mL tubes at 37°C for 24 hours while providing shaking is needed. LifeCanvas’s EasyClear is very useful for this step in the SHIELD protocol.
It takes 4-5 days to preserve mouse organs, and it is worth the wait! SHIELD samples produce more meaningful data through better preservation of antigenicity and fluorescence, along with allowing multiplexing of labeling.
Store the SHIELD-Epoxy Solution and SHIELD-ON Buffer at 4°C. SHIELD-Buffer Solution may be stored at room temperature. The shelf life is approximately 2 months.
It should be disposed of along with PFA. This will usually be handled by your institution’s internal waste disposal service.
Stochastic electrotransport technology allows rapid lipid clearing and saves significant time. A whole mouse brain can be cleared in 5-7 days instead of needing at least 3-4 weeks when cleared passively.
SmartClear II Pro is a turn-key solution and is much easier, safer, and more reliable than any homemade setup. The standardization of our system means that you can start generating data earlier, with less troubleshooting and wasted resources. Your results will also be more consistent and quantitative with standardized hardware in use by a rapidly growing customer base.
Our proprietary buffer composition ensures that over the entire span of buffer lifetime (~10 days) pH is maintained at a level that doesn’t disrupt endogenous fluorescent proteins and cause tissue deformation. SmartClear II Pro’s membranes prevent black precipitate buildup (a byproduct of SDS) on the electrode surface from transferring to the tissue itself.
DIY systems work for thinner slices, but significant tissue deformation can occur with whole-brain clearing due to the strong electric fields common in DIY setups. These setups would also lack our patented rotational technology, which has been optimized to ensure even clearing of tissue while not causing damage.
We have expertise in preservation, clearing, labeling and imaging, so we can we can provide guidance and troubleshooting for your experiments. This is something no other company or lab can offer.
Many of our users clear mouse brains. However, users have cleared spinal cord, liver, tumor, lung, heart, kidney, and bladder before, as well as brains from additional species such as rat and bird (zebra finch) and human samples. As more people adopt the system, the variety of sample types and sizes will increase. We also help with troubleshooting/optimizing protocols for specific tissue samples, and have holders available for samples of many different sizes. Our standard sample holder is quite flexible and can accommodate many types/sizes/shapes of tissues.
No, it is not. However, SHIELD-preserved tissue is very robust, and therefore allows many rounds of staining and de-staining without losing antigenicity and suffering structural deformation.
There is slight tissue expansion, but it will usually shrink back down to normal size after index matching.
Theoretically, if the sample can fit inside the clearing chamber, it can be cleared. However, larger tissue samples will take more time to clear. Rat brains can take around 6-8 days to clear and even larger samples may take prohibitively long, so blocking the tissue into sections is recommended to speed clearing (if it’s compatible with your research goals).
Imaging quality will be impacted if samples become too thick in either the XY (lateral) or Z/axial dimension, so this should be a consideration when designing experiments.
SmartClear II Pro uses a dual-buffer and membrane system. The membranes separate the electrodes from the clearing chamber. This prevents the build-up of black precipitate on the samples. To keep the membranes hydrated, one buffer is only pumped through the clearing chamber (buffer A) and the other buffer is only circulated around the electrodes on the opposite side of the membrane (buffer B).
Buffers last for up to 10 days. This 10 day lifetime only applies when the clearing power is turned on. You can save buffers and membranes for later use.
We have optimized buffer composition to achieve this 10 day lifetime to maintain pH and buffer volume. It is normal for the reservoir volume of Buffer A to increase by up to ~150 mL during the first several days of each new membrane + buffer pair’s 10-day lifetime, due to electroosmotic flow of Buffer B across the membrane. This does not affect clearing quality or speed and will reverse itself during the latter half of the 10-day lifetime.
It depends on the tissue type, but the Refractive Index is usually about 1.46.
The warranty of the system is only valid if our consumables are used. We have optimized buffer composition for the best clearing performance and lifetime. We cannot guarantee this performance if other consumables are used.
No, SmartClear II Pro is for SDS-based aqueous clearing methods only. It is compatible with tissues preserved with SHIELD, CLARITY, or SWITCH. Samples that have only been perfused & post-fixed with PFA, and which have not been through one of the preservation protocols mentioned here, are not suitable for clearing. PFA-only samples lose their fluorescence and often undergo severe structural deformation and loss of antigenicity upon delipidation.
If you have a unique sample, we can make a custom holder for you. The mesh bags are also customizable with a simple impulse sealer.
If the electrodes wear out during the length of the warranty, we will replace them. SmartClear II Pro comes with a 1-year warranty with the option of purchasing a warranty extension.
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