Is it possible to over-clear your tissue?

I'm clearing whole brains and spinal cords from adult mice, passively. It seems to be going well so far- it's been about a month and there is definitely clearing happening. My question has to do with whether it is possible to "over"clear tissue with CLARITY. In my samples, the clearing isn't necessarily even across the tissue; obviously with regard to depth the outer regions will clear faster than the middle, but there are also a few areas that seem to have minimal clearing in a spinal cord that is almost entirely clear elsewhere (possibly because I didn't remove the dura). Should I be worried about leaving those mostly-cleared samples at 37C for the time it takes for those scattered areas to become clear? (Does that risk any damage to the areas that have already cleared?) Thanks!


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    We’re late to contribute an answer to your question but better late than never, right? Yes, it’s possible to over-clear your tissue. It definitely shouldn’t take a whole month to clear a brain or a spinal cord - at least not in our experience using our ClearLight reagents. Remember, that tissues will not necessarily look clear to the naked eye depending on the tissue type and structure. You can have a tissue be CLARITY clear without it looking visually clear. The CLARITY technique is just removing light scattering lipids, not other components. The spinal cord will not appear completely clear during the clearing process. It’s composed of multiple components besides lipids. On our Tissue Clearing Comparison page, you can see how tissues that have been cleared will look to the naked eye and then see how they look after immunostaining and imaging following the RI match. I hope this helps.
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