The overall goal of these psPACT and mPACT methods is the fast clearing of whole CNS tissue without the need for an electrophoretic tissue clearing system. This method can help answer key questions in the human disease research model field, such as Alzheimer's disease or injury and degeneration and tumorigenesis. The main advantage of this technique is that the mouse CNS tissue is not damaged by the electrophoretic tissue clearing, and it's rapidly cleared by modified passive clearing.
We first had the idea for this method after using the original PACT and past method for brain clearing. Visual demonstration of this method is critical as psPACT and mPACT method are difficult to run because this method has multiple stages of sample preparation. Begin by preparing PACT cocktail solution by adding VA-044 powder to a solution of 4%PFA and 4%acrylamide at a final concentration of 0.25%After perfusing the tissues with PACT solution, completely immerse the whole brain and spinal cord into a 50-milliliter tube containing chilled PACT solution, and store at four degrees Celsius for 24 hours.
24 hours later, transfer the tissue to a 50-milliliter tube containing fresh, cold PACT solution, and connect the tube cap to a tissue gel hybridization system, connected to a nitrogen tank and set to 37 degrees Celsius. Then, turn on the vacuum and gas for 10 minutes to embed the sample in nitrogen gas. To polymerize the hydrogel, place the tube containing the sample in a shaking incubator set to 150 rpm and 37 degrees Celsius for three hours or until polymerization is complete.
Following the incubation, use blotting paper to remove the remaining polymerized hydrogel surrounding the tissues. Then, transfer the tissue to a 50-milliliter tube containing clearing solution. Place the sample in a shaking incubator set to 37 degrees Celsius and 150 rpm until the tissue has been cleared.
On average, it takes about 20 days for the mouse CNS to achieve full clarity. Begin psPACT with the CNS from a mouse perfused with 4%PFA that has been post-fixed in 4%PFA at four degrees Celsius for 24 hours. Wash the fixed tissue for one hour in 0.1 molar PBS.
After washing, transfer the tissue to A4P0 solution, and incubate at 37 degrees Celsius for 24 hours. The next day, after washing the tissue in PBS for five minutes, immerse the tissue in 0.25%VA-044 in PBS, and incubate at 37 degrees Celsius for six to 24 hours. Separating the acrylamide and subsequent VA-044 step avoids the need for removing remaining unpolymerized hydrogel monomers that surround the tissue, which can compromise final tissue integrity after achieving transparency with PACT.
Following the incubation, transfer the tissues to fresh, cold VA-044 solution. Then, embed the sample in nitrogen gas for 10 minutes. Transfer the tissue to clearing solution.
Then, incubate the sample in a shaking incubator set to 37 degrees Celsius and 150 rpm until the tissue has been cleared. On average, it takes approximately 17 days for mouse CNS to achieve full clarity. For mPACT, follow the protocol for psPACT until the clearing solution step.
Then, fully immerse the tissue clearing solution containing 0.5%alpha-thioglycerol, an un-browning agent, in addition to 8%SDS in 0.1 molar PBS. Place the sample in a shaking incubator set to 37 degrees Celsius and 150 rpm until the tissue has been cleared. On average, it takes only two weeks for the mouse CNS to achieve full clarity using mPACT.
In the present study, mouse brain samples treated via the mPACT protocol achieved optical transparency after 14 days, ahead of samples treated via the PACT and psPACT protocols. Rat whole CNS samples cleared by mPACT achieved maximal clearance in 20 days. Adult rat brains alone, as opposed to the entire CNS, were cleared with mPACT in a mere five days.
Once mastered, this technique can be done in two weeks with the mouse whole CNS if it is performed properly. After its development, this technique paved the way for researchers in the field of neuroscience and anatomy to explore pathophysiologic disease, such as tissue injury, developmental malformations, cancer, and neurodegenerative disease in mammalian CNS models.
