hello Debaprasad Koner it depends whether you isolated your mitochondria intact. Most of available dyes depend on the presence of the membrane potential of mitochondria. You need to extract them in an MP preserving medium to stain them with rhodamine 123, if you wish to fix them and then view them microscopically mitotracker dyes retain well after fixation. Specifics of these dyes can be found here (https://www.thermofisher.com/kw/en/home/references/molecular-probes-the-handbook/probes-for-organelles/probes-for-mitochondria.html). Method for isolating MP maintained mitos are many but this reference is a classic (PMID: 11381591), or (Article Isolation of Intact Mitochondria from Skeletal Muscle by Dif...

Thank you Rabeah Al-Temaimi for your kind response. I have isolated mitochondria and kept in respective buffer. After adding Mitored, i checked under confocal microscope. I m getting signals also. But, i cant understand whether I m on correct track or not!!!