Here is the link to the protocol, on which we have based our approach; however we have made a few alterations: https://docs.abcam.com/pdf/protocols/clarity-protocol.pdf
The hydrogel embedding and custom-built ETC whole-brain tissue-clearing parts of the protocol have shown successful results; however, when imaging the clarified samples either with light-sheet or confocal microscopy, the fluorophore (e.g.; monosynaptic GFP-Rabies virus, transgenic mice) seems to have been completely quenched.
The following parameters are used:
Constant current of 1.5A which stabilizes around 55V
Temperature alternating between 30˚C and 40˚C
pH level beginning around 8.5 and dropping to 7.3 within two to three hours
The following links show the custom built setup:
**Note: The filter component has been replaced with a 60mL Nalgene container, in which the SDS solution (1L H2Omq, 200mM SDS, 20mM lithium hydroxide monohydrate, and 0.5% 1-thioglycerol) passes through a Falcon cell strainer before reaching the sample. Secondly, the reservoir has been replaced with a 1L Nalgene container.
1. I have been using a constant 1.5A and 1L of solution as suggested by (Lee, et al. 2016; DOI: 10.1038/srep18631); however, in papers such as (Kim, et al 2018; DOI:10.1038/s41598-018-31153-7) and (Kim, et al 2018; DOI:10.1038/s41598-018-26776-9) it lists the better parameter as 1.5mA. Is this a typo?
2. It has been demonstrated that using platinum plates, instead of platinum wires, leads to a slower decrease in clearing solution pH level. Does anyone know a vendor who sells platinum plates?
3. Since we have seen a continuing quenching of the fluorophore after active clarification, what are the main parameters in the active CLARITY process that could be contributing to fluorophore quenching? Should we consider adding Bis-Acrylamide into the hydrogel monomer solution to induce a stronger cross-linking? Should we consider leaving the active method, and switch to PaCT?
Thank you for your time and help! I'd be happy to provide any further information.