Hi fellow researchers. I am facing some problems with the mitochondrial staining hence seeking for your advice. I had tried several times now trying to stain mitochondria using mitotracker deep red with several concentrations. I am trying to observe the mitochondria network if it's of your concern. I am no expert in imaging and this is my first time conducting such an experiment. I followed every step as specified in the protocol provided by the company (Invitrogen) but I don't think the acquired image gave a good representation of the mitochondrial network. I am using HEK293T cell (GFP transfected). A brief protocol as conducted:
1) 50 ug of lyophilized MitoTracker Deep Red was diluted in 100 ul of DMSO to produce 0.92mM stock (kept in -20°C)
2) 100 nM and 200 nM working solution was prepared, diluted in Opti-MEM
3) Incubated with the respective concentration for 30 minutes in 37°C
4) Cells were washed with Opti-MEM (1x) before fixed with 4% PFA in PBS for 15 minutes
5) Fixated cells were washed with PBS (3x)
6) Observed under FV-1000 (Olympus)
Attached is some of the image I got if needed. Much appreciated if anyone can share their experience so that I can do some troubleshooting regarding the staining.
Hi, if you are using chemical dyes like mitotracker, or lysotracker, you should not have fixed your cells with PFA or methanol. Those fixatives will perforate those organelles and lead to diffusive staining.
Another thing you need to pay attention to is the exitation wave length. Why are you using "deep red"? I don't know which type of microscope you are using for this imaging.Are you sure that it's compatible with your microscope?
Thank you for the suggestion Siwei... The protocol did stated that fixation can be done with 4% PFA.... Hence, followed as stated... With the problem contributed by using PFA, will you give advice on any fixative compound I can use to fix my cell without/lessen the probability of the probe from diffusing? thank you...
I am using deep red because the stained mitochondria will retain even after fixation as certain mitotracker did not... I am aware that certain other mitotracker stain better after fixation... However as my laboratory already possessed deep red, so im just make use of it... Im using FV-1000 (olympus) to acquire those images... The imager did not exactly built with the specific wavelength of interest but we adjust the wavelength to be as close as possible... Im not sure if it is specifically compatible or otherwise... Can you advice me regarding this matter?
Thank you for the kind respond, Siwei...
I would recommend you confirm your microscope can excite and detect the far red fluorophore before you troubleshoot too much more, but I would say the next step would be to try:
30min incubation @ 300nm
You can also adjust the incubation time to fit your needs, since you are fixing the cells post-treatment, it may be necessary to use a higher concentration to make sure you retain enough of the signal.
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