Loss of mCherry fluorescence in fixed samples

[George Campbell]

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Hello all,

I am trying to image mCherry as a transfection marker in cells cultured on
coverslips that I immunostain for other proteins. However, I have noticed a
decrease in signal when comparing my live and fixed samples.

Fixation method: 4% PFA
Mounting material: ProLong Gold

Do you think that either the chemical fixation or the mounting medium is
affecting mCherry?

Also, can anyone vouch for an mCherry antibody that works well in
immunofluorescence studies?

Thanks,
George

[Linda Barthel]

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George,
This is odd.  In our samples the mCherry is very resistant to chemical
fixation, we routinely fix in 4% paraformaldehyde.  It is also very stable
when exposed to hot sodium citrate antigen unmasking.  Gibco/Invitrogen now
carries a new rat monoclonal antibody specific for mCherry.
Good luck,
Linda


On Thu, Jun 27, 2013 at 1:38 PM, George Campbell <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello all,
>
> I am trying to image mCherry as a transfection marker in cells cultured on
> coverslips that I immunostain for other proteins. However, I have noticed a
> decrease in signal when comparing my live and fixed samples.
>
> Fixation method: 4% PFA
> Mounting material: ProLong Gold
>
> Do you think that either the chemical fixation or the mounting medium is
> affecting mCherry?
>
> Also, can anyone vouch for an mCherry antibody that works well in
> immunofluorescence studies?
>
> Thanks,
> George
>

--
Linda Barthel, M.S.
*Research Laboratory Specialist Lead*

*Department of Molecular, Cellular, and Developmental Biology*
                      3010 Natural Sciences Building (Kraus)
                                    830 N. University
                             Ann Arbor, MI  48109-1048
lab: [phone]
fax: [phone]
http://www-personal.umich.edu/~praymond/

      *  Microscopy & Image-analysis Laboratory-North *
            Biomedical Research Core Facilities
              2800 Plymouth Rd, Rm 53S, Bdg 20
                 Ann Arbor, MI  48109-2800

office: [phone]
fax:    [phone]
http://www.umncrc.org

[Mark A. Sanders]

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Hi George,

 I agree with Linda that mCherry fluorescence retention (80+%) is not typically a problem if fixed correctly, but will add that the pH of the working solution of the 4% FRESH paraformaldehdye is critical and should be 7.2-7.4 for vertebrate cells.  Check the working solution with pH strips and adjust with 0.1N NaOH to the correct pH.  

I have used ProLong Gold with mCherry successfully.  Be sure to let it cure to optimal R.I.

Cheers,
Mark
****************************************************
Mark A. Sanders      University of Minnesota
Program Director      Twin Cities Campus
University Imaging Centers      
St. Paul office ph:  612-624-3454
Mpls office ph:  612-626-3645
fax: [phone]
www.uic.umn.edu

 

On Jun 27, 2013, at 1:29 PM, Linda Barthel <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> George,
> This is odd.  In our samples the mCherry is very resistant to chemical
> fixation, we routinely fix in 4% paraformaldehyde.  It is also very stable
> when exposed to hot sodium citrate antigen unmasking.  Gibco/Invitrogen now
> carries a new rat monoclonal antibody specific for mCherry.
> Good luck,
> Linda
>
>
> On Thu, Jun 27, 2013 at 1:38 PM, George Campbell <
> [hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello all,
>>
>> I am trying to image mCherry as a transfection marker in cells cultured on
>> coverslips that I immunostain for other proteins. However, I have noticed a
>> decrease in signal when comparing my live and fixed samples.
>>
>> Fixation method: 4% PFA
>> Mounting material: ProLong Gold
>>
>> Do you think that either the chemical fixation or the mounting medium is
>> affecting mCherry?
>>
>> Also, can anyone vouch for an mCherry antibody that works well in
>> immunofluorescence studies?
>>
>> Thanks,
>> George
>>
>
>
>
> --
> Linda Barthel, M.S.
> *Research Laboratory Specialist Lead*
>
> *Department of Molecular, Cellular, and Developmental Biology*
>                      3010 Natural Sciences Building (Kraus)
>                                    830 N. University
>                             Ann Arbor, MI  48109-1048
> lab: [phone]
> fax: [phone]
> http://www-personal.umich.edu/~praymond/
>
>      *  Microscopy & Image-analysis Laboratory-North *
>            Biomedical Research Core Facilities
>              2800 Plymouth Rd, Rm 53S, Bdg 20
>                 Ann Arbor, MI  48109-2800
>
> office: [phone]
> fax:    [phone]
> http://www.umncrc.org

[SUBSCRIBE CONFOCALMICROSCOPY Sara Smith]

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Hi George,

You may want to use the freshly prepared PFA. We had experienced GFP
fluorescence loss when using old PFA.


Yi


On Thu, Jun 27, 2013 at 4:07 PM, Mark Sanders <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi George,
>
>  I agree with Linda that mCherry fluorescence retention (80+%) is not
> typically a problem if fixed correctly, but will add that the pH of the
> working solution of the 4% FRESH paraformaldehdye is critical and should be
> 7.2-7.4 for vertebrate cells.  Check the working solution with pH strips
> and adjust with 0.1N NaOH to the correct pH.
>
> I have used ProLong Gold with mCherry successfully.  Be sure to let it
> cure to optimal R.I.
>
> Cheers,
> Mark
> ****************************************************
> Mark A. Sanders      University of Minnesota
> Program Director      Twin Cities Campus
> University Imaging Centers
> St. Paul office ph:  612-624-3454
> Mpls office ph:  612-626-3645
> fax: [phone]
> www.uic.umn.edu
>
>
>
> On Jun 27, 2013, at 1:29 PM, Linda Barthel <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > George,
> > This is odd.  In our samples the mCherry is very resistant to chemical
> > fixation, we routinely fix in 4% paraformaldehyde.  It is also very
> stable
> > when exposed to hot sodium citrate antigen unmasking.  Gibco/Invitrogen
> now
> > carries a new rat monoclonal antibody specific for mCherry.
> > Good luck,
> > Linda
> >
> >
> > On Thu, Jun 27, 2013 at 1:38 PM, George Campbell <
> > [hidden email]> wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Hello all,
> >>
> >> I am trying to image mCherry as a transfection marker in cells cultured
> on
> >> coverslips that I immunostain for other proteins. However, I have
> noticed a
> >> decrease in signal when comparing my live and fixed samples.
> >>
> >> Fixation method: 4% PFA
> >> Mounting material: ProLong Gold
> >>
> >> Do you think that either the chemical fixation or the mounting medium is
> >> affecting mCherry?
> >>
> >> Also, can anyone vouch for an mCherry antibody that works well in
> >> immunofluorescence studies?
> >>
> >> Thanks,
> >> George
> >>
> >
> >
> >
> > --
> > Linda Barthel, M.S.
> > *Research Laboratory Specialist Lead*
> >
> > *Department of Molecular, Cellular, and Developmental Biology*
> >                      3010 Natural Sciences Building (Kraus)
> >                                    830 N. University
> >                             Ann Arbor, MI  48109-1048
> > lab: [phone]
> > fax: [phone]
> > http://www-personal.umich.edu/~praymond/
> >
> >      *  Microscopy & Image-analysis Laboratory-North *
> >            Biomedical Research Core Facilities
> >              2800 Plymouth Rd, Rm 53S, Bdg 20
> >                 Ann Arbor, MI  48109-2800
> >
> > office: [phone]
> > fax:    [phone]
> > http://www.umncrc.org
>