labeling lipids in water contineous emulsions

[Martin Koster]

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For years, we have been using Nile Blue A (ex. Janssen Pharmaceutics) to label both lipids, and proteins of oil in water emulsions. It was simple to prepare a 1% aqueous solution; 1 gram 1 of Nile Blue A in 100 ml demineralised water, and the solution ended up to be a mix of Nile Blue and a small amount of Nile Red (equilibrium). Nile Blue for the proteins, and Nile Red for the fat/lipids.

Excitation at 488 yields emission around 520-630 for the lipids, and excitation at 633 has emission in the range 660-750. As the 488 signal bleeds into the 633 we do sequential imaging.

But we ran out of supply of the NB powder. Since the original supplier does not have this in its portfolio anymore we bought a small batch from Sigma Aldrich. However, we now have issues not experienced before. Very low signal, especially for the lipids indicates this powder does not dissolve as easy as the old powder, and we get almost no Nile Red in the solution.
After researching this a bit, it was found that heating up and acidifying with sulphuric acid helps in dissolving. After cooling/filtering it is now usable for a few weeks, but then the dye starts precipitating again, and we get less bright images.

Questions:
-Has anyone a good preparation protocol for Nile Blue A (to get a mix of Nile Blue and Nile Red in water). We need an aqueous solution, because the emulsions are water continuous.
-Is there a good alternative to Nile Red for labelling fat/lipids (which does hardly dissolve in water)?

[George McNamara]

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Hi Martin,

Molecular Probes / ThermoFisher has DiI and a large number of related
fluorophores. Used in a different context (blood vessel painting), Yiwen
Li and Rong Wen published a DiI protocol using freshly made solution in
glucose:

Direct labeling and visualization of blood vessels with lipophilic
carbocyanine dye*DiI*. <https://www.ncbi.nlm.nih.gov/pubmed/18846097>

*Li Y*, Song Y, Zhao L, Gaidosh G, Laties AM,*Wen R*.

Nat Protoc. 2008;3(11):1703-8. doi: 10.1038/nprot.2008.172.

PMID:
    18846097


there may be a little more information in:

Low magnification confocal microscopy of tumor angiogenesis.
<https://www.ncbi.nlm.nih.gov/pubmed/24052350>

McNamara G, Yanai A, Khankaldyyan V, Laug WE, Boden J, Webster K,*Li
Y*,*Wen R*.

Methods Mol Biol. 2014;1075:149-75. doi: 10.1007/978-1-60761-847-8_6.
Erratum in: Methods Mol Biol. 2014;1075: E1-3.

PMID:
    24052350


Yiwen and Ron like the Sigma-Aldrich DiI because $0.50 per mouse with
their 2008 protocol. You might want higher purity grade. The Di_ family
are available in lots of tail lengths and colors (DiA, DiO, DiI, DiD,
DiR). A couple of other companies sell similar fluorophores, such as the
Claret series from Sigma-Aldrich.


enjoy,

George


On 1/10/2017 3:41 AM, Martin Koster wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> For years, we have been using Nile Blue A (ex. Janssen Pharmaceutics) to label both lipids, and proteins of oil in water emulsions. It was simple to prepare a 1% aqueous solution; 1 gram 1 of Nile Blue A in 100 ml demineralised water, and the solution ended up to be a mix of Nile Blue and a small amount of Nile Red (equilibrium). Nile Blue for the proteins, and Nile Red for the fat/lipids.
>
> Excitation at 488 yields emission around 520-630 for the lipids, and excitation at 633 has emission in the range 660-750. As the 488 signal bleeds into the 633 we do sequential imaging.
>
> But we ran out of supply of the NB powder. Since the original supplier does not have this in its portfolio anymore we bought a small batch from Sigma Aldrich. However, we now have issues not experienced before. Very low signal, especially for the lipids indicates this powder does not dissolve as easy as the old powder, and we get almost no Nile Red in the solution.
> After researching this a bit, it was found that heating up and acidifying with sulphuric acid helps in dissolving. After cooling/filtering it is now usable for a few weeks, but then the dye starts precipitating again, and we get less bright images.
>
> Questions:
> -Has anyone a good preparation protocol for Nile Blue A (to get a mix of Nile Blue and Nile Red in water). We need an aqueous solution, because the emulsions are water continuous.
> -Is there a good alternative to Nile Red for labelling fat/lipids (which does hardly dissolve in water)?

--


George McNamara, PhD
Houston, TX 77054
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75/
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650