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When comparing brains processed by our modified method or the original protocol, we found our modifications permit faster and more efficient clearing and labeling. Our studies would allow high-content tracing and analysis of intact brain or other large-scale samples in a short time. For centuries, analysis of large-scale tissues mostly depended on microtomes to cut thin sections and provide cellular information at a two-dimensional level.

However, biological specimens are intrinsically three-dimensional 3D , so lots of information is lost when cutting them into sections. Therefore, interrogating biological samples at the 3D level is necessary. Combined with various labeling methods, tissue clearing methodologies permit the interrogation of molecular, cellular and system biology across different scales. It transforms tissue into a hydrogel-tissue hybridization and preserves proteins and nucleic acids at their position but eliminates lipids with sodium dodecyl sulfate SDS solution to achieve tissue optical transparency 3 , 4.

Compared with other tissue clearing techniques, CLARITY and its variants have high tissue compatibility and have been successfully used in most organs of the mouse, the whole body of mouse and post-mortem human brain tissue 5 , 6 , 7 , 8. Further, CLARITY-processed tissues permit several rounds of labeling as proteins and nucleic acids are bound to hydrogel network by chemical bonds while other tissue clearing techniques cannot 2.

First, the electrophoresis system is complex so it is difficult to build and maintain. In the clearing system, many devices are needed sample chamber, chemical resistant tube, refrigerated circulator, filter and peristaltic pump and the system must be leak-proof. And it builds a closed circulation system, but the circulation does not always work stably as it is easily interrupted by bubbles which form continually during electrophoresis. These two main problems impede its application in wider fields. In general, many variants focused on two aspects: 1 simplifying the procedures and modifying the system to suit different specimens; and 2 shortening the processing time.

As for lipid extraction, the key procedure of tissue clearing, CLARITY and its variants are based on two methods: electrophoresis and passive thermal diffusion. Electrophoretic lipid extraction is faster than passive thermal diffusion, but it requires building a circulation system while passive diffusion does not, which makes it difficult to manipulate.

The principle of the staining process is similar to lipid extraction, so it shares problems. Therefore, it is important to find an optimized way that can both shorten the processing time and simplify the system for easy manipulation. We designed a non-circulation electrophoresis system NCES to perform electrophoretic lipid extraction. This system is much simpler to build than that mentioned in the original protocol, and it permits multiple sample clearing at the same time.

In addition, we made modifications to the hydrogel embedding methods, clearing buffer and immunostaining method. Our study not only simplifies electrophoretic tissue clearing but also saves much time so that the method can be applied in more studies. To simplify the electrophoresis system, we removed the circulation, stored the SDS clearing buffer in a beaker and performed electrophoresis in the beaker Fig.

Furthermore, we designed a mobile electrophoresis device that allowed observing samples easily during electrophoresis by lifting it.

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It is also easy to clean byproducts that adhere to electrodes. The electrophoresis device and sample chamber were made by a common PMMA processing plant, and it is less expensive less than 10 dollars than using lots of devices including electrophoresis chamber, peristaltic pump, and filter to set a complex circulation system hundreds of dollars.

Moreover, the electrophoresis device and sample chamber could be designed in different sizes and structures to suit different specimens and achieve multiple sample clearing at the same time we tested clearing two brains at the same time.


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Starting clearing of several samples simultaneously by modifying the electrophoresis device and sample chamber could greatly improve clearing efficiency. Additionally, as the SDS clearing buffer was stored in the beaker, bubbles forming during electrophoresis would float to the water surface and dissolve gradually.

In this way, the electrophoresis current would not be interrupted by bubbles. As for byproducts forming during electrophoresis, we noted that they always deposited on the mouse surfaces next to the electrodes. So we hypothesized that most of the byproducts were charged molecules. We set qualitative filter paper in the two sides next to the electrodes, and this prevented byproduct deposition. The real object and detailed dimensions are provided in Supplementary Fig. In our modified methods, we adopted A4P0B0 to perform hydrogel embedding A4P0B0-processed brain was the experimental group.

In electrophoresis clearing, the A4P4B0. The cleared brain could not be imaged with a confocal microscope. The transmittance of the A4P4B0. The image of the A4P0B0-processed brain also showed clear neuronal axons and dendrites Fig. It demonstrated our modifications including A4P0B0 embedding method and adding a-thioglycerol could achieve faster clearing and higher transparency than the A4P4B0.

Error bars in all histograms denote standard deviation. Images of electrophoresis cleared A4P0B0-processed brain. It shows dopaminergic neurons stained with Anti-Tyrosine Hydroxylase antibody at a part of the midbrain. It was reconstructed with the filament auto-path toolkit in Imaris 9. Moreover, the clearing speed was different when using different PFA post-fixation times.

The post-fixation time is usually determined empirically. Thus we explored the effect of different post-fixation times on clearing speed. The brain expanded and became more homogeneous with less opacity when using shorter time post-fixations. This showed that the clearing speed was highly related to PFA post-fixation time. In addition, the brain became yellow easily during electrophoresis because of the Maillard reaction.

As for passive clearing, both the A4P4B0. However, the transmittance of the A4P4B0. However, the A4P4B0.

Furthermore, the A4P4B0. Therefore, our modified A4P0B0-processed passive brain clearing not only resulted in faster clearing speed but also achieved transparency at a mild temperature to avoid autofluorescence bleaching. It is a projection of image stacks and shows stained dopaminergic neurons in the passive cleared A4P4B0. Images of passive cleared A4P0B0-processed brain.

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Dopaminergic neurons stained with Anti-Tyrosine Hydroxylase antibody. It is reconstructed with the filament auto-path toolkit in Imaris 9. In passive clearing, it is hard to achieve transparency in the central area of the brain. Generally, the brain quickly becomes transparent except for the central area, and extended times are needed to clear this area.

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First, we hypothesized that it was because the distance between this area and the clearing buffer is greater than other parts of the brain, and the clearing buffer did not easily reach the central part as the surrounding structure obstructed its penetration. So we set the passive clearing with a 2-mm brain A4P0B0-processed coronal slice of the central part. In the brain slice, this area was directly contacted by clearing buffer, and the distance between each brain part and the clearing buffer is the same.

However, though the brain slice was almost cleared after 4 days of clearing, the central area in accordance with the less transparent area of intact brain was still opaque Supplementary Fig. So it is not the distance that makes the central area hard to clear. Moreover, we found that the brain would expand and become homogeneous after a certain duration of electrophoresis while the brain processing passive clearing not Supplementary Fig.

Then we tried to perform a short time electrophoresis before passive clearing and interrogate whether the electrophoresis processing could promote the next passive clearing.

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Both the A4P4B0. After electrophoresis, the brains expanded and became homogeneous but were still opaque Fig. After passive clearing, both the A4P4B0. For the A4P4B0. The transmittance of electrophoresis cleared A4P4B0.