Using LiBH4 stock in THF

[HaamsRee]

Has anyone tried diluting LiBH4 with water from a 2 M stock in THF? Sigma sells it in 25 mL aliquots. I'm trying to set up the protocol for high throughput, and weighing the powder is a bit of a pain😔

[radtkea]

Hello, thank you so much for asking your question in the open! I haven't tried it, but I think I know some colleagues who might have...I will get back to you. Exciting news about the high throughput. Fingers crossed.

[HaamsRee]

Thanks for looking into this. Any updates?

[radtkea]

Thank you so much for the follow up, HaamsRee. I am still waiting for confirmation. I did look into JoVE articles and other published protocols by IBEX members. Everyone uses powder from what I can see in the materials/methods:

Powder: https://app.jove.com/t/67869/three-dimensional-imaging-tumor-bearing-tissue-using-iterative
Powder: https://app.jove.com/t/67853/iterative-bleaching-extends-multiplicity-with-use-staining-automation
Powder: https://app.jove.com/t/68644/high-plex-imaging-using-spectral-confocal-microscopy-to-minimize-non

I cannot access this article, but emailed the author: https://app.jove.com/v/67696/high-throughput-imaging-analysis-workflow-for-evaluating-skin-cell

I understand the frustrations with weighing out the powder. I recommend sticking with this protocol until further notice. Best of luck with your research. Would love to connect and learn more, if you have time/interest.

[zivy]

Hello @ HaamsRee,

This is outside my wheelhouse so I asked colleagues and got the response: “We haven't tried it so we cannot recommend it.”

As an aside I was told that “this also requires evaluation of safety procedures with the chemical supplier, the institute, etc.”, so be careful.

Finally, if you decide to go down this novel path, please share your experience. If it is successful, then I expect we will read about it in the literature. Even if it doesn’t work out, it would be great if you shared information with the community on what you tried and what didn’t work. Possibly a post to this discussion. This will allow others to follow in your footsteps and attempt to overcome the issues.

Best of luck with your research.

[CarolCKoh]

In our lab, we are using Lithium borohydride solution, 2 M in THF (Sigma, UN3399-4.3, 4×25 mL).

We take a small amount from the stock solution using an insulin syringe and needle and transfer it to an Eppendorf tube. The “small amount” is the minimum we can withdraw, sometimes up to 100 μL. From this Eppendorf, we then take 11.5 μL to prepare a 1 mg/mL solution in water by adding it to 500 μL of water (giving a final volume of ~511.5 μL), which we use immediately (we allow ~10 minutes for bubble formation). We do not store this solution.

The 2 M stock bottle has a self-sealing septum cap, similar to those used for vaccines, which allows the needle to penetrate while keeping the bottle closed and sealed. When we transfer the solution to an Eppendorf, we observed that, even when kept protected from light in the storage box, the stock reacts over time and becomes dark. Therefore, we also discard the remaining stock in the Eppendorf after use. For this reason, we always try to take the smallest possible volume from the original stock using the insulin syringe.

Preparation and application of 1 mg/mL LiBH4 solution:

Allow the solution to incubate at room temperature for ~10 minutes, until bubble formation is observed.

Add 50–150 μL of the LiBH4 solution to the tissue (depending on the size of the section) and incubate for 30 minutes at room temperature, exposing it to white light.

Every 10 minutes, we remove the LiBH4 from the tissue and replace it with fresh solution.

This modification improved the bleaching outcome compared to our previous protocol, which used only 15 minutes of incubation.

[radtkea]

Thank you so much, CarolCKoh! This is tremendously helpful!