Q: What kind of staining reagents can be used?
A: a) Antibodies: direct fluorescent-labeled antibodies are preferred. For example, the antibodies are diluted adequately in PBS containing both 0.5% Triton™ X-100 and 0.01% NaN3.
b) Nuclei staining reagents: propidium iodide can be used. Propidium iodide is diluted to 10 µg/mL with 0.1 M phosphate buffer (pH 7.4), containing 0.5 M NaCl.
Q: Can antibodies from my laboratory be used?
A: Some proteins do not change their antigenicity during fixation or tissue-clearing procedures. However, this is not conformed for all the proteins. It is recommended to initially test the antibodies you are already using.
Q: Can fluorescent-labeled secondary antibodies be used?
A: We do not have information about protocols using secondary antibodies. It takes significant amounts of time for the two-step treatment with both primary and secondary antibodies. Therefore, we recommend directly labeling your primary antibody with fluorescent reagents.
Q: What kind of fluorescent proteins can be used?
A: We assessed the retention of fluorescence intensity in GFP. Some fluorescent proteins, such as, EGFP, EYFP, mCherry, and mKate2, have been confirmed to retain their fluorescence signals (Cell 2014, 157, 726.)
Q: What kind of fluorescent labeling dyes can be used for immunohistochemical staining during clearing step?
A: It is confirmed in references (Cell 2014, 159, 911. or Cell Rep. 2018, 24, 2196.) that Such fluorescent labeling dyes as FITC, Rhodamine, Alexa594 and Alexa647 can be used.
Q: What kind of container is suitable?
A: Any container that is slightly larger than the organism specimen being cleared is suitable. For mouse organ clearing, a container that is slightly bigger than the organ itself is recommended because the organ may expand during the clearing procedure. CUBIC products are aqueous-based reagents, thus they can be used safely with any laboratory plasticware such as polypropylene or polyethylene.
Q: Does tissue swell? If yes, will this influence the experiment in any way?
A: The tissue or organs may expand during the clearing; However, it has been reported that relative cell position remains the same. Thus, expansion is linear and uniform.
Q: Could the sample fixation step be omitted because the organs undergo the clearing steps as soon as they are excised?
A: In the samples without fixation, the structure of cells may be destroyed, thus, they should be fixed before clearing steps.
Q: Is it possible to clear the samples after they were excised and fixed?
A: The samples which were soaked in fixing solution for several weeks or which are stored at -80°C for several months after fixation can still get cleared.
Q: Can paraffin embedded samples be sectioned and get cleared by CUBIC?
A: Paraffin embedded samples can get cleared by CUBIC after thermal deparaffinization. However, samples should not be sectioned to a few µm thick. After getting cleared, samples become fragile and a few µm thick of samples cannot be treated. Therefore, samples should be sectioned by razor or something to one mm thick or thicker and then treated under CUBIC. The reference (Sci. Rep. 2017, 7, 9269.) mentions the way to clear samples in detail.
Q: What quantity of these reagents are required for tissue clearing?
A: For mouse whole-body clearing, the volume of reagents used must be sufficient to submerge the entire specimen. For example, for the whole-body clearing of a mouse, 200 to 400 mL of CUBIC-L and 100 to 200 mL of CUBIC-R+ are needed. For mouse tissue clearing, the volume of reagents needed is half the volume of the organ being cleared. For example, 20 to 40 mL of CUBIC-L and 10 to 20 mL CUBIC-R+ are needed.
Q: The clearing of organs or bodies was interrupted and did not occur successfully.
A: There are a few possible reasons. Consider the following troubleshooting options.
a) The pH of PFA solution for fixing organs or bodies is too high.
When the pH is more than 8, organs and bodies become over-fixed and are less cleared, thus, the pH should be adjusted between 7 – 7.5.
b) Delipidation is incomplete.
Samples are immersed in CUBIC-L with gentle shaking at 37 °C for 2 – 5 days or more, and fresh CUBIC-L should be used daily.
c) Clearing is incomplete.
The clearing time period can be extended. Additionally, consider replacing and using a fresh CUBIC-R+ solution.
Q: How long does it take to delipidate samples?
A: Approximately 3 days are required to delipidate the lung, intestine, pancreas and spleen of an adult mouse, and approximately 5 days to delipidate the heart, brain, liver and kidney.
Q: Mounting Solutions (Product Number: M3294 or M3292) are necessary for observation cleared samples?
A: If the observation is temporary, samples can be observed in not mounting solutions but CUBIC-R+ or CUBIC-X2 with the samples and objective lens soaked. However, when it takes for a long time (more than one hour) to get images or something, the CUBIC reagents evaporate during the observation because they contain water. Evaporation may cause the refractive index change and make image acquisition difficult, and as evaporation proceeds, the CUBIC reagent may crystallize. Therefore, it is recommended to use mounting solutions which contain no water for long-term observation.
Q: How should the reagents be disposed of following use?
A: Please dispose of the reagents according to the regulations of your institution. Reagents used to soak animal or organ samples are typically treated as medical waste. The unused CUBIC-L, and R+ reagents are non-flammable waste liquids. Please refer to the included package insert for reagent descriptions and constituencies.
Q: How should clearing samples be stored?
A: Clearing samples can be stored at room temperature in CUBIC-R+ or CUBIC-X2. CUBIC-R+ and CUBIC-X2 contain many solutes and a little water as a solvent. Thus, the samples should be stored sealed by parafilm or other means to prevent reagents from evaporation. The agarose gel embedding sample can also be stored at room temperature.
Q: Clearing samples cannot be observed well.
A: Light-sheet fluorescent microscopy (LSFM) or confocal laser-scanning microscopy (CLSM) is recommended for the observation of the samples. Clearing samples become gel-like and it may be difficult to cut them into thin slices. The samples should be observed in Mounting Solution (RI=1.520) or Mounting Solution (RI=1.467) with objective lens which are available of these RIs.
Q: What is the refractive index (RI) of CUBIC reagents?
A: The RI of CUBIC-R+ is 1.52 and that of CUBIC-X2 is 1.467. The objective lens or the immersion oils which are suitable for these RIs should be used. They should not be mixed with other solvents such as water in order to change their RIs.
Q: CUBIC-1, CUBIC-2 have been described in some papers, are they the same as CUBIC-L, CUBIC-R+?
A: CUBIC-1, CUBIC-2 differ from CUBIC-L, CUBIC-R+ in terms of their clearing ability, as CUBIC-L, CUBIC-R+ is superior. CUBIC-1 and CUBIC-L play the same role in delipidation and decoloring, and CUBIC-2 and CUBIC-R+ play the same role in RI matching. CUBIC-R also differs from CUBIC-R+. CUBIC-R is composed of nicotinamide and CUBIC-R+ is composed of N-methylnicotinamide. CUBIC-R+ is superior to CUBIC-R in terms of maintaining fluorescent signals.
**The clearing or staining result differ according to the samples or staining reagents. Please examine the treatment time or the concentration of staining reagents.