Here you will find commonly asked questions and their solution. If you have further questions please contact us on This email address is being protected from spambots. You need JavaScript enabled to view it. and we will be happy to answer them.

LABELING

  • [➕] 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.
  • [➕] uDISCO seems to preserve GFP very well. How about conventional fluorescent dyes that stain nucleus? Such as DAPI, Hoechst?
  • [➕] Which florecent protein shall I use best?
  • [➕] 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?
  • [➕] I wanted to try to first clear the brain and then do immunostaining on it. Would this be possible?
  • [➕] Have you tried NeuN staining with your cleared samples and could you please share your staining protocol for my reference?

CLEARING

  • [➕] I saw the online protocol on your website (please find attached screenshot) and I didn't understand the meaning of "ON".
  • [➕] 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).
  • [➕] 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.
  • [➕] 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?
  • [➕] Can uDISCO be used to image cells/structures within bone?
  • [➕] Have you worked on brain tumors? If yes, is it fine for clearing experiments?
  • [➕] Could uDISCO cleared brains be put into fluid with refractive index close to water?
  • [➕] In uDISCO, which chemicals cause the most quenching, and does pH affect this as well?
  • [➕] What is the reason for fluorescence bleach caused by 3DISCO?
  • [➕] Do you have any advice on the ration of BABB to DPE? Also, is your lightsheet objective an immersion lens?
  • [➕] How about incredibly fatty tissues like mesentery?
  • [➕] As the tissue needs to be cleared with (and kept in) BABB, we wondered if for imaging this caused any safety issues?
  • [➕] 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?

IMAGING

  • [➕] Does the shrinkage impact your ability to resolve fine structures?
  • [➕] We are going to perfuse a GFP mice. Can you suggest how long can I keep the cleared brain in DBE without affecting the fluorescence? I can only image it on Monday and I was planning to complete the clearing on Friday and keep the tissue in DBE until Monday.
  • [➕] Once the tissue is cleared should the tissue be stored in DBE or any other solution? If it is stored in DBE will it quench the fluorescent signals? We don't have immediate access to a microscope in our facility, so I would like to know if I can store it for a longer time (4-5 days) in solution and then image it without compromising on the fluorescence?