Hello, I am curious about the reasoning behind some of the differences between the 2016 iDISCO and the 2023 rat-iDISCO protocols regarding tissue processing:
Why are many of the dehydration, bleaching, and clearing steps in the rat-iDISCO protocol performed rocking as o
Read moreEmily Jane Dennis Answered Nov 25, 2024
Janelia Research Campus
Hello, thanks for your interest in our protocol.
We empirically made many decisions across many variables, and this was the most successful, consistent, and concise protocol we came up with... however, we did not try every permutation of every step and there may be other global maxima in the landscape of cleared brains. It is possible that rocking is not required, for example, we just always rocked all brains and therefore we report that here. We were inspired by conversations with others, both the iDISCO and uDISCO protocols, as well as our Laura's experience and expertise (Laura Lynch, one of the co-authors, has cleared thousands of brains across many species).
However, the extension of the time in methanol gradient washes was the largest positive effect on clearing and we feel is absolutely crucial based on our extensive but not exhaustive empirical exploration of the chemical space.
Re: perfusion. I have several samples from fieldwork where perfusion is not possible and can speak to this with experience. In general, if one does not perfuse the brain, one will have bright autofluorescence of much of the vasculature. The utility of these brains is up to the user. For some uses, having capillaries/etc visible is not a problem. For others, especially cell counting from tracing or staining, the capillaries interfere with automated analyses that are critical to use for these types of large volume data. So, the brains will not fully clear without perfusion, but that may or may not be a concern depending on one's experimental needs. This is not unique to our protocol but is true for effectively all clearing methods.
Best of luck and hope this is helpful,
Emily
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