Free of acrylamide sodium dodecyl sulphate (SDS)‐based tissue clearing (FASTClear): a novel protocol of tissue clearing for three‐dimensional visualization of human brain tissues

Does the Sleep-Related Neurons Modulate the Sensation of Pain under the Use of GA?

“…These different GA are used to judge whether it is extensive of a specific phenomenon in several GA. As for the first part, the experiment begins with the Fos TVA gene knock-in mice during the embryo state. Then GA and pain stimulation are applied to the mice according to their assigned groups and GA. After the mice are sacrificed and stored at 4˚C, the mice brain will be processed with Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY), which is a tissue clearing technology that can transform intact biological tissue into a three-dimensional transparent hydrogel-based structure [12], and will be examined through confocal microscopy. Pain activated neurons can be found and ensured after observation.…”

“…Tissue-hYdrogel (CLARITY), tissue clearing technology, is an advanced technique that can transform intact biological tissue into a three-dimensional transparent hydrogel-based structure with all its essential structures [12]. After CLARITY operation, the membrane-bound GFP and Fos are still in place be-cause of the hydrogel skeleton support.…”

“…Hydrogel polymers have to be grown from inside the tissue to support the mice's brain's structure and molecular content. This can be done with the infusing of 4˚C hydrogel monomers cocktail, formaldehyde, and thermally triggered initiators into the tissue [12]. Formaldehyde serves the dual purposes of cross-linking amine-containing tissue components to each other and covalently binding the hydrogel monomers to native biomolecules including proteins, nucleic acids, and other small molecules but not a membrane.…”

“…Formaldehyde serves the dual purposes of cross-linking amine-containing tissue components to each other and covalently binding the hydrogel monomers to native biomolecules including proteins, nucleic acids, and other small molecules but not a membrane. Then, the hydrogel polymerization is triggered by the heat of 37˚C [12]. Because of the formaldehyde binding effect, lipids can now be extracted from the brain without destroying important components of the brain.…”

Vasculature-staining with lipophilic dyes in tissue-cleared brains assessed by deep learning

“…Blood flow is important in basic research as well as in pathology such as stroke, and the reconstruction of the vasculature in three-dimensions (3D) is a valuable investigative tool. Within the last decade several tissue-clearing techniques, have been developed, which make large samples transparent and enables microscopy of the 3D structures within intact organs (Ertürk et al, 2012;Chung et al, 2013;Yang et al, 2014;Epp et al, 2015;Zheng and Rinaman, 2016;Kolesová et al, 2016;Neckel et al, 2016;Liu et al, 2017;Carrillo et al, 2018;Qi et al, 2019;Cai et al, 2019). One of the early, and still, powerful methods is CLARITY (Clear Lipidexchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel) (Gradinaru et al, 2018;Jensen and Berg, 2017), which utilizes an acrylamide-hydrogel to keep proteins bonded in a skeleton structure and the removal of lipids, which are the primary cause of light-scattering in tissues, by using a detergent leaving the tissue transparent (Chung et al, 2013;Tomer et al, 2014).…”