Why Aren’t My Tissues Clearing?

Retinal Organoid

A retinal organoid cleared using the Visikol HISTO clearing technique and imaged using confocal microscopy at 10X magnification. DAPI labeling is clearly visible throughout the entirety of the organoid.

You have decided to take on whole mount labeling of a tissue or in vitro generated spheroid/organoid, and the labeling itself looks good, but you cannot seem to clear all the way through the tissue. The tips and tricks here are specifically related to Visikol HISTO clearing techniques but can likely be applied to other solvent-based clearing techniques as well. The goal of tissue clearing is to be able to make an entire tissue structure translucent by matching the refractive index of the tissue to the surrounding solution. Ultimately, there is only one reason that a tissue didn’t clear all the way and that is because the refractive index isn’t uniform throughout the tissue.

How can this be fixed?

Firstly, remember that size correlates with time- it will take longer for a thicker sample to become clear than for a smaller sample. Make sure you don’t skimp out on your dehydration gradient, it is important to the process to

  • Do a gradual gradient, as it helps equilibrate the sample and removes water more efficiently, it also has the added benefit of minimizing potential shrinkage.
  • Make sure the sample is in the dehydration solutions for the specified amount of time, see the Visikol Guidebook for more detailed information.

Specific to Visikol HISTO clearing, some tissues may be able to be cleared just using Visikol HISTO-1 or Visikol HISTO-M (organoids/spheroids), but often, use of Visikol HISTO-2 provides substantial improvement. If using Visikol HISTO-2, always do dehydration gradient, then Visikol HISTO-1, then Visikol HISTO-2.

If you’ve followed the guidebook recommendations and still aren’t seeing clearing, there are a few possibilities:

  • The ethanol or methanol being used has absorbed water from the air, resulting in water not being fully removed, and thus cloudiness in the sample.
  • The sample vessel was not sealed properly, and the hygroscopic Visikol HISTO-2 is drawing in water.
  • Insufficient volume of Visikol HISTO-1, Visikol HISTO-2, or Visikol HISTO-M for your tissue size. Using insufficient volume of Visikol HISTO reagents can cause inadequate clearing.
  • Make sure you have not made the mistake of using Visikol HISTO-2 in an incompatible plastic dish, which can result in plastic leaching into the sample affecting clearing.

Conveniently, if you are using the Visikol HISTO clearing technique, the clearing process is reversible so, if it seems like you haven’t gotten all the water out of the sample, you can always reverse steps and try again. If you need additional assistance with clearing your samples, please feel free to reach out! We offer both technical assistance for our products as well as contract services, such as whole mount labeling, tissue clearing and advanced imaging.

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