MitoTracker Red and Fixation

[Claire Brown]

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Hi All,

 

We are trying to fix some cells with MitoTracker Red. We usually use it live but were trying to do some photobleaching experiments. The dye seems to be quenching or washing out when we fix the cells. We tried using methanol free PFA and fixing at 37oC as the Probes protocol suggests.

 

Any suggestions?

 

Sincerely,

 

Claire

 

 

[Iain Johnson]

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Aldehyde fixation will certainly cause destaining because it kills the mitochondrial membrane potential that drives the electrophoretic uptake of the dye. Any dye that isn't coupled to protein will wash out.  

To maximize the protein coupling (which is thiol, not aldehyde, mediated), you want to make the mitochondrial interior as reduced as it can possibly be before you do the PFA. I used to use a quick 1 mM TCEP treatment step for this.

Iain

Sent from my iPhone

On Sep 27, 2016, at 4:09 PM, Claire Brown, Dr. <[hidden email]> wrote:

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Hi All,

 

We are trying to fix some cells with MitoTracker Red. We usually use it live but were trying to do some photobleaching experiments. The dye seems to be quenching or washing out when we fix the cells. We tried using methanol free PFA and fixing at 37oC as the Probes protocol suggests.

 

Any suggestions?

 

Sincerely,

 

Claire

 

 

[Konstantín Levitskiy]

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Hi.

 

About 10 years ago we bought MitoTrackerDeepRed (MTDR) and we had some use in vivo. When we fixed we noticed something different. We expressed pEYFP-Mito in cells and after checking total colocalization with a dye we washed with a PFA, seeing how the dye was internalized in another place but with an aspect “similar to the mitochondria”. No was colocalization at all after fixing. We asked about it to Molecular probes, but with no answer.

 

Regards,

Dr. Konstantín Levitskiy

Servicio de Microscopía

InstitutodeBiomedicinadeSevilla - IBiS

Campus del Hospital Universitario Virgen del Rocío

Avda. Manuel Siurot s/nº

41013 Sevilla

Tlfno: 955 92 3030

Email: [hidden email]

Web: www.ibis-sevilla.es

 

 

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---

De: Confocal Microscopy List [mailto:[hidden email]] En nombre de Iain Johnson
Enviado el: martes, 27 de septiembre de 2016 19:14
Para: [hidden email]
Asunto: Re: MitoTracker Red and Fixation

 

Aldehyde fixation will certainly cause destaining because it kills the mitochondrial membrane potential that drives the electrophoretic uptake of the dye. Any dye that isn't coupled to protein will wash out.  

To maximize the protein coupling (which is thiol, not aldehyde, mediated), you want to make the mitochondrial interior as reduced as it can possibly be before you do the PFA. I used to use a quick 1 mM TCEP treatment step for this.

 

Iain

 

Sent from my iPhone

On Sep 27, 2016, at 4:09 PM, Claire Brown, Dr. <[hidden email]> 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. *****

Hi All,

 

We are trying to fix some cells with MitoTracker Red. We usually use it live but were trying to do some photobleaching experiments. The dye seems to be quenching or washing out when we fix the cells. We tried using methanol free PFA and fixing at 37oC as the Probes protocol suggests.

 

Any suggestions?

 

Sincerely,

 

Claire