Hello
I am working on Drosophila testis and would like to use Rhodamine 123 to visualize mitochondria in the live tissue samples. I've ordered Rhodamine 123 from Sigma and received a vile with purplish powder without any data sheets. I found on the internet that this material is soluble in ethanol, and has been reported soluble in ether, various fats and lipids, DMSO, and has limited solubility in water (i guess in PBS as well). The problem is that I would like to stain whole live testis and ethanol or ether will kill the tissue for sure. Should I dissolve the powder in ethanol or DMSO to make a high concentration stock solution and then dilute it in PBS to make a working solution? Or maybe it will affect the permeability of the material?
Thank you in advance for your help!
I would suggest to get TMRE. It is a derivative of Rh123 but more lipophilic. It is soluble in DMSO and has better chances to get and equilibrate in tissue.
Something that has worked for me in the past is dimethyl formamide. It will address your solubility and viability needs.
Thank you for your advice.
Sergei, I already got Rh123, so I would like to try it first and if it won't work I will look for alternative.
Michael, I am wondering if dimethyl formamide won't damage the tissue? I personally used formamide for fixation in the past...
High conc stock in EtOH is quite stable. Try to make a rh123 titration assesing by Facs to determine the final conc of rh123 for your system. Include a 1-5 microM of FCCP as control. Avoid fixing.
What is the final concentration you are trying to achieve in your incubation buffer? The amt of DMF in the final buffer will likely not fix anything.
so you propose to make a stock solution in DMF and then dilute it in lets say PBS for staining?
I already diluted it to 10mg/ml in DMSO for the stock solution and 10ug/ml in PBS for the working solution and it worked very well. Thanks everybody for the help!
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