Abstract
Purpose :
Membrane-permeant cationic fluorescent dyes like tetramethylrhodamine ethyl ester (TMRE), equilibrate across membranes in a Nernstian fashion; thus, accumulating into the mitochondrial membrane matrix space in inverse proportion to Ψm and are predominantly employed to monitor this membrane potential in cells. However, the conventional dye loading methods used for staining poses some limitations in the form of non-specific loading, high signal to noise ratio and fluorescence toxicity, which compromises the precision of the results; especially in tissue slices mainly due to the thickness of the neuronal layer which augments background fluorescence.
Methods :
Using a McIlwain tissue slicer, we have taken 150 mm slices from adult rat retina and focally loaded it with 500 nM TMRE using a pressure injector at 5 psi, angled at 30 degree from the surface of the tissue for 5-10 minutes. In another sets of experiment we bath loaded the slices with 50nM TMRE. To measure the signal to noise ratio in both focal and bath loaded retina slices with TMRE, the tissue was exposed to the mitochondrial membrane uncoupler, 500μM CCCP.
Results :
The slices loaded with our improvised methadology showed significantly increased signal to noise ratio and minimum non-specific background fluorescence when compared with the conventional bath loading method.
Conclusions :
The improvised focal loading of mitochondrial dye introduces an alternate way to image the mitochondria using minimal dye with maximum output intensity so as to increase the precision of the results. Our methodology can also be extended to other tissues and dyes.
This is a 2020 ARVO Annual Meeting abstract.