LifeCanvas FAQs

Below you will find answers to most commonly asked questions regarding LifeCanvas’s products and technology. Don’t see your query? Contact us at info@lifecanvastech.com and we will be happy to share our expertise. 

SHIELD

What does the acronym stand for?

It stands for Stabilization to Harsh conditions via Intramolecular Epoxide Linkages to prevent Degradation.

What does SHIELD do?

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. 

What is the SHIELD protocol?

You can find the full length protocol here. 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.

What are the advantages of SHIELD over CLARITY?

  • Protein preservation is greatly enhanced, especially the native fluorescence of endogenous fluorescent proteins
  • There is significantly less sample to sample variability via a simpler process that requires minimal optimization for different sample types.
  • Cleared samples are not as fragile – less chance of physical damage.
  • It allows for multi-round destaining and restaining for multiplexed imaging, as the sample is exceptionally well-preserved to withstand the high temperatures (70°C) needed.

Why should I use SHIELD over other tissue preservation methods?

  • SHIELD greatly enhances protein preservation, especially the native fluorescence of endogenous fluorescent proteins.  
  • Allows destaining, restaining and multiplexed imaging.
  • Does not quench tdTomato.
  • Far less autofluorescence with SHIELD.
  • SHIELD preserves mRNAs for detection using fluorescence in situ hybridization (FISH).

Do I need to perfuse? Can I post-fix samples?

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.

Do I need to do a degassing step?

No, there is no degassing step required.

Is there autofluorescence?

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.

What tissues can I preserve?

Any tissues that can be preserved with CLARITY can also be preserved with SHIELD. This includes brain, liver, lung, heart, kidney, pancreas, skin, etc.

Which animals is SHIELD compatible with?

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.

Can I use FISH on SHIELD-preserved tissues?

Yes, SHIELD preserves mRNAs for detection using fluorescence in situ hybridization (FISH).

How much does it cost?

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.

How many samples can I preserve with each kit?

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).

Do I need to buy a machine to use SHIELD?

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.

How long does it take?

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.

How is it stored, and what is the shelf life?

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.

How do I dispose of SHIELD solutions?

It should be disposed of along with PFA. This will usually be handled by your institution’s internal waste disposal service.

Is it toxic to work with? Are there any special handling considerations?

Like all lab chemicals, SHIELD should be handled with the appropriate personal protective equipment. Gloves, lab coat, and eye protection are recommended. The MSDS sheets for each SHIELD solution are linked below. 

SHIELD-Epoxy Solution MSDS

SHIELD-Buffer Solution MSDS

SHIELD-ON Buffer MSDS

SmartClear II Pro

Why should I buy SmartClear II Pro instead of using CLARITY to clear tissues passively?

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.

Why should I buy SmartClear II Pro instead of making my own system?

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.

Why should I use SmartClear II Pro over other detergent based or organic clearing methods?

  • The clearing is more homogenous due to the rotating electric field.
  • There is less chance of even microscopic sample deformation due to our patent pending stochastic electrotransport technology.
  • SmartClear II Pro is more convenient, only requiring buffer changes once every 10 days instead of every day or every other day. This also means you only use 1 L of buffer every 10 days, for less shipping, waste, and release of detergents into wastewater systems.
  • SmartClear II Pro’s membranes keep black SDS precipitate away from the tissue surface.
  • Our company was founded by the inventor of CLARITY and we have unparalleled technical support. We can help with every step of tissue processing, including preservation, clearing, labeling and imaging. We are the first company to offer such a pipeline and as such have unique experience.

What tissue types can be cleared?

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.

Is the clearing reversible?

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.

Is there tissue expansion or contraction?

There is slight tissue expansion, but it will usually shrink back down to normal size after index matching.

How large tissue samples can be cleared?

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.

How does the buffer system work?

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.

What is the Refractive Index (RI) of the cleared tissue?

It depends on the tissue type, but the Refractive Index is usually about 1.46.

Can I make and use my own clearing buffers?

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.

Is SmartClear II Pro compatible with iDISCO?

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.

Do you have custom sample holders?

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.

Will I have to replace the electrodes?

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.