- [➕] Why do you perfuse the mice for 30 min and if it is essential to perfuse for 30 min? Usually, we perfuse it with saline for 3 min and then 8-9 min with 4% PFA.
30min is just suggested time. We perfuse for 5-10 min with PBS to wash the blood out, this is very important if you want to decrease the background and not to see all the vessels that can disturb your imaging. I usually perfuse for 20 min with 4% PFA because normally this makes the body hard and well fixed after this amount of time. You can of course decrease the duration of the PFA perfusion for special cases.
- [➕] uDISCO seems to preserve GFP very well. How about conventional fluorescent dyes that stain nucleus? Such as DAPI, Hoechst?
So far we observed that the following nucleus staining work very well with uDISCO: DAPI, Hoechst, Propidium iodide, TO-PRO3. SYTO16 was not compatible though.
- [➕] Which florecent protein shall I use best?
In general every fluorescent protein works well. However, we do not suggest tdTomato, which somehow is weak in DISCO clearing.
- [➕] In your paper you used YFP in the uDISCO routine. Do you know if the proteins (eYFP and YFP) would work similarly well or is there again a bleaching issue to be expected?
Both (eYFP or YFP) are expected to survive the uDISCO and give you strong signal.
- [➕] I wanted to try to first clear the brain and then do immunostaining on it. Would this be possible?
No, unfortunately it is not possible. The clearing removes the water from tissue, while antibodies work in a water environment incubation solution and cleared tissue cannot be placed back in water or PBS. We are currently working on the rehidration procedure though…
- [➕] Have you tried NeuN staining with your cleared samples and could you please share your staining protocol for my reference?
For staining protocol at the moment I would suggest iDISCO clearing paper where they use 3DISCO as tissue clearing: PMID 2541716
- [➕] I saw the online protocol on your website (please find attached screenshot) and I didn't understand the meaning of "ON".
This means overnight.
- [➕] Is it necessary to keep the brain/tissue for post-fixation in PFA overnight or can we proceed directly with the clearing? Is it possible to post-fix the brain in PFA and keep it at -80 until clearing (maybe a few days later).
We usually keep brains overnight in post fixation in PFA to make sure that brains are well fixed, you can try to clear them immediately after the perfusion and see the result. We never tried to keep brains at -80.
- [➕] With my last clearing, the brain (left hemisphere) was not very clear. I changed the DBE frequently but till the end it didn't clear although the brain shrunk and it became stiff. In another instance, the brain became transparent but it was light brown in color. Kindly, suggest your opinions on how can I improve these problems.
It is absolutely normal that everytime brains and organs shrink, this because our tissue clearing protocol removes water from tissues and they decrease of size, and it is also normal that they become stiff and with brownish colour due to chemical reactions. Be sure to put your sample in adequate time, you can increase the incubation time of each step and see if the clearing can improve or not.
- [➕] We are having trouble in being able to use tert-butanol in our lab as it is highly flammable. Our EHS department is not happy with us using it. How do you deal with this compound?
Tert-butanol is indeed highly inflammable, therefore we treat it according to our safety regulations. First of all the stock solutions are always stored in fire-resistant cabinets. However, since tert-butanol is solid below 25-26 °C, we slightly heat it up at 37°C using an incubator just immediately prior to use, being sure that the bottles are well sealed and that temperature never goes over 37°C. When it starts melting then it is ready to be used as pure or diluted with distilled water. Once diluted, it will not solidify anymore at room temperature and you can keep it in the fire-resistant cabinets. All the clearing protocols, included the whole body clearing with the perfusion pump, are done inside a fume hood to prevent the clearing solution to contaminate the lab environment.
- [➕] Can uDISCO be used to image cells/structures within bone?
Yes, uDISCO can be used to image the structures in the bones. uDISCO renders whole bones transparent, allowing visualization of inner bone structures. However, the background signal in the bones are usually high. Therefore, we recommend usage of labeling in far-red range.
- [➕] Have you worked on brain tumors? If yes, is it fine for clearing experiments?
uDISCO and 3DISCO clearing can be used for visualizing any cell type in the brain, and therefore it can be potentially employed to visualize brain tumors.
- [➕] Could uDISCO cleared brains be put into fluid with refractive index close to water?
No. The refractive index of untreated brain is around 1.37-1.41 (PMID: 22274454
) and this will increase to about 1.56 after clearing because of shrinkage. The cleared brains will turn to be non-transparent if immersing into refractive-index-mismatch reagents (e.g. RI_water = 1.33).
- [➕] In uDISCO, which chemicals cause the most quenching, and does pH affect this as well?
Fluorescence quenching is affected by several factors, including pH, and also by the properties of fluorescent structures. For example, YFP is very sensitive to acidic pH and lose approximately 50% of its fluorescence at pH 6.5. Wild-type GFP is also quenched by acidic pH values with an apparent pKa near 4.5. EYFP is sensitive to chloride ions and bleaches much more than GFP. Compared with diphenyl ether, tert-butanol, benzyl alcohol and benzyl benzoate would reduce fluorescence to some extent since the active hydroxyl, carbonyl groups.
- [➕] What is the reason for fluorescence bleach caused by 3DISCO?
Fluorescence bleaching is affected by several factors, including pH, and also by the properties of fluorescent structures. For example, YFP is very sensitive to acidic pH and lose approximately 50% of its fluorescence at pH 6.5. Wild-type GFP is also quenched by acidic pH values with an apparent pKa near 4.5. EYFP is sensitive to chloride ions and bleaches much more than GFP.
- [➕] Do you have any advice on the ration of BABB to DPE? Also, is your lightsheet objective an immersion lens?
For the ratio between BABB and DPE, one can play around between 4:1 and 15:1, keeping in mind that the more DPE you add, more preserved the signal will be, but less cleared the sample is.
- [➕] How about incredibly fatty tissues like mesentery?
Since the fatty tissues are mainly composed by lipids and most protocols (organic / water solvent based clearing) are trying to remove them in order to increase the penetration of IHC, clearing and RI matching reagents. Both 3DISCO and uDISCO are using DCM for delipidation and thus are not very suitable for fatty tissues.
- [➕] As the tissue needs to be cleared with (and kept in) BABB, we wondered if for imaging this caused any safety issues?
Regarding the BABB clearance, we do not do much actually. We have good ventilation, and also keep the windows open. LaVision might have suggestions from other users too.
- [➕] I would like to understand how should I prepare the tissue after clearing for visualizing it on a microscope. Is it necessary to have the sample in DBE and then visualize it or can I just place the tissue, put a coverslip and image it?
The sample must be completely immersed in DBE. If you have a light sheet microscope, the system should already be optimized for clearing solution, but CHECK IT before dipping any objective inside the clearing solution.
If you have a confocal microscope you have to build a close chamber where to put your sample and then fill it with DBE (using a histology glass, silicon glue or dental cement and a coverslip), because normally confocal objective are not meant to be dipped in DBE. Be aware that DBE can potentially melt the glue and/or components of the microscope and the objectives.