Issue with Fluorophore Quenching
stephen_james
Posts: 1
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:
Setup_FrontView: https://drive.google.com/file/d/1n0vf5S9_CYRD-SgXB2cFFb2Ifqf-Do2a/view?usp=sharing
Setup_TopView: https://drive.google.com/file/d/13ktnk1liz6k54j39Cxe-_ON0x3bNhQyd/view?usp=sharing
Setup_SideView: https://drive.google.com/file/d/19fXu5x7-C4ughs40r5XbKhv1kSN0JksM/view?usp=sharing
**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.
Questions:
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.
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:
Setup_FrontView: https://drive.google.com/file/d/1n0vf5S9_CYRD-SgXB2cFFb2Ifqf-Do2a/view?usp=sharing
Setup_TopView: https://drive.google.com/file/d/13ktnk1liz6k54j39Cxe-_ON0x3bNhQyd/view?usp=sharing
Setup_SideView: https://drive.google.com/file/d/19fXu5x7-C4ughs40r5XbKhv1kSN0JksM/view?usp=sharing
**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.
Questions:
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.