ECi-based tissue clearing

Ethyl-3-phenylprop-2-enoate (ethyl cinnamate, ECi) is an organic solvent with a refractive index of 1.558. ECi is a Food and Drug Administration and European directive 67/548/EWG approved food flavor and additive for cosmetic products. According to this, it is non-toxic and can be handled without specific safety measures. (For the original publication, see: Klingberg et al. Fully Automated Evaluation of Total Glomerular Number and Capillary Tuft Size in Nephritic Kidneys Using Lightsheet Microscopy.)

Frequently asked questions (FAQ) regarding ECi-based tissue clearing

Here, we collect frequently asked questions (FAQ), which we received from scientists working with ECi to clear their samples. If you have a question regarding our ECi clearing, do not hesitate to contact Prof. Dr. Matthias Gunzer.

1. General Remarks on ECi Clearing

  • Q: How are the cleared samples stored?
    ECi-cleared samples should be stored in ECi at room temperature in the dark. ECi freezes at 6-8°C, so don’t put your samples in the fridge! Additonally, mind that ECi is not compatible with polystyrene vessels. Use polypropylene or glass vessels! Furthermore, optical clearing is a reversible effect. If you put your samples back into EtOH-solutions, they will become opaque again.

2. Dehydration (Pre-Clearing)

  • Q: Do I have to use EtOH for the sample dehydration to be able to use ECi clearing?
    NO, you can use anything (THF, MeOH) that draws the water out of your sample sufficiently. You can even clear the sample following your normal procedure in e.g. DBE and transfer it afterwards to ECi for imaging. Beware that the RI is matching your sample after your protocol is completed!
  • Q: How do I adjust the pH of the ethanol series?
    For adjusting the pH to a basic level (about 9.0), 0.1M NaOH and 0.1 HCl are used. As the pH is related to water-based solutions only, the pH levels of the water containing EtOH solutions (50% EtOH and 70% EtOH, not 100% EtOH) are adjusted. For pH calibration, indicator strips as well as pH electrodes can be used
  • Q: Is the pH of the ethanol series stable? Do I need an exact pH = 9?
    Yes, the adjusted pH is stable for several weeks if you consider the following aspects: According to Le Chatelier’s principle the temperature is affecting the pH-equilibrium of the solutions massively. Hence all steps of pH adjustment and tissue dehydration should be performed at constant temperature (4-8°C). Meaning, the ethanol solutions should be cooled down to 4-8°C before pH adjustment and they should be stored at 4-8°C. Furthermore, Aqua ad iniectabilia not distilled water should be used for generating the 50% and 70% EtOH solutions. Distilled water tends to absorb carbon dioxides from the atmosphere and thereby becomes acidic very fast.
    No, it is not necessary to adjust the pH exactly to 9.0, other basic pH levels (pH 8-11) stabilize the endogenous fluorescent proteins too.

3. Clearing with ECi

  • Q: Could you please tell me at what concentration ECi should be applied? In what solution is it solved?
    ECi can be used as it is provided by the supplier as a pure liquid. You do not need to solve it in anything else. Beware of freezing in the fridge!

4. Post-Processing

  • Q: Can cleared samples still be used for histological analysis?
    Yes, they can. As the clearing is reversible, you can rehydrate your samples and use them for histology. Here, the ECi is washed out of the tissue via incubation in decreasing EtOH solutions (100%, 70% and 50%) containing a permeabilizing agent (Tween20/Triton X100). After tissue rehydration, the samples can be used for paraffin or cryo-sectioning. IHC as well as IF stainings are still working.

5. Organ-specific Questions

  • Q: Can I clear lipid rich structures like brain with ECi?
    Yes, you can. During the dehydration process you need to incorporate delipidation. In the published protocol, we use 2% Tween20 added to the ethanol. If this is too weak for you, just use THF, methanol or DCM, which have high delipidation qualities. Beware of toxicity and handle with care.
  • Q: How long does it take to clear bone tissue?
    4 h PFA, 12h each of 50%/70% and 2x 12 h in 100% Ethanol, 6h clearing in ECi (Klingberg et al. Fully Automated Evaluation of Total Glomerular Number and Capillary Tuft Size in Nephritic Kidneys Using Lightsheet Microscopy., suppl. Table 2)
  • Q: Do you have experience using the clearing protocol on bones of large animals, e.g. minipigs?
    We do not work with larger animals. It should principally be possible, but it will take far longer.
  • Q: Is it possible to stain bone tissue with fluorescent antibodies following clearing with ethyl cinnamate?
    We stain after fixation, but before tissue dehydration (EtOH series) and refractive index matching (ECi.). It is possible, but takes very long. Everything you can stain inside of a live animal is much faster and more complete.
  • Q: For the lungs, did you remove the agarose they were inflated with (and if so, at what point?) or was this left in?
    The agarose stays in the lung and is dehydrated and cleared together with the rest of the organ.