Imaging GFP with BrdU

[Claude Messier]

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Although this is not a confocal question proper, I would appreciate
suggestions for a immunohistochemistry problem. We are trying to visualize
GFP in the brain of transgenic mice expressing GFP in oligodendrocyte
precursor cells. In order to identify dividing oligodendrocyte precursor cells,
we also want to use BrdU labelling. The problem is that the acid treatment
that is needed to obtain BrdU staining also abolish the GFP visualization. GFP
staining is not extremely strong to start with so we have used anti-GFP
secondaries to improve visualization. We have manipulated several variables
associated with the acid treatment (length, concentration, heat, pH) or
fixation (mostly length of fixation) to no avail. This is a common problem
with BrdU since the acid treatment interferes with some antibodies. Although
we have used the EdU click-it system successfully, we are testing a large
number of animals and the cost of using EdU is significant if not prohibitive.
So, any suggestion to obtain BrdU staining while preserving endogenous GFP
stain?
Many thanks!
Claude

[Katrin roth]

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Dear Claude,

we also used EdU click-it system. To minimalize the EdU usage we used 50ml Falcons in the drinking bottles and we generated little agarose pads with the EdU which is feeded.

All the best
Katrin
________________________________________
Von: Confocal Microscopy List [[hidden email]] im Auftrag von Claude Messier [[hidden email]]
Gesendet: Dienstag, 28. Mai 2013 14:31
An: [hidden email]
Betreff: Imaging GFP with BrdU

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Although this is not a confocal question proper, I would appreciate
suggestions for a immunohistochemistry problem. We are trying to visualize
GFP in the brain of transgenic mice expressing GFP in oligodendrocyte
precursor cells. In order to identify dividing oligodendrocyte precursor cells,
we also want to use BrdU labelling. The problem is that the acid treatment
that is needed to obtain BrdU staining also abolish the GFP visualization. GFP
staining is not extremely strong to start with so we have used anti-GFP
secondaries to improve visualization. We have manipulated several variables
associated with the acid treatment (length, concentration, heat, pH) or
fixation (mostly length of fixation) to no avail. This is a common problem
with BrdU since the acid treatment interferes with some antibodies. Although
we have used the EdU click-it system successfully, we are testing a large
number of animals and the cost of using EdU is significant if not prohibitive.
So, any suggestion to obtain BrdU staining while preserving endogenous GFP
stain?
Many thanks!
Claude

[Linda Barthel]

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Claude,
What normality is your HCl?  We have used BrdU in combination with eGFP
with much success.  Our HCl is 2N in PBS with 0.5% triton.  Our treatment
for cryosections is for 1/2 hr followed by 2 buffer rinses and then proceed
with our ICC as usual.
Regards,
Linda Barthel

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



On Tue, May 28, 2013 at 8:31 AM, Claude Messier <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Although this is not a confocal question proper, I would appreciate
> suggestions for a immunohistochemistry problem. We are trying to visualize
> GFP in the brain of transgenic mice expressing GFP in oligodendrocyte
> precursor cells. In order to identify dividing oligodendrocyte precursor
> cells,
> we also want to use BrdU labelling. The problem is that the acid treatment
> that is needed to obtain BrdU staining also abolish the GFP visualization.
> GFP
> staining is not extremely strong to start with so we have used anti-GFP
> secondaries to improve visualization. We have manipulated several variables
> associated with the acid treatment (length, concentration, heat, pH) or
> fixation (mostly length of fixation) to no avail. This is a common problem
> with BrdU since the acid treatment interferes with some antibodies.
> Although
> we have used the EdU click-it system successfully, we are testing a large
> number of animals and the cost of using EdU is significant if not
> prohibitive.
> So, any suggestion to obtain BrdU staining while preserving endogenous GFP
> stain?
> Many thanks!
> Claude
>

--
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

[George McNamara]

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Hi Claude,

1. anti-GFP with tyramide signal amplification (another Invitrogen
product, also available from PerkinElmer in fewer colors) ... another
not necessarily cheap reagent, but can be optimized for both volume and
amount used. Also can buy in bulk.

2. If you order a Click-iT EdU in bulk you can get a discount (bigger
quantities, bigger discount). Could save you a lot of time=money than
dealing with the many variables you wrote about.


George


On 5/28/2013 7:31 AM, Claude Messier wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Although this is not a confocal question proper, I would appreciate
> suggestions for a immunohistochemistry problem. We are trying to visualize
> GFP in the brain of transgenic mice expressing GFP in oligodendrocyte
> precursor cells. In order to identify dividing oligodendrocyte precursor cells,
> we also want to use BrdU labelling. The problem is that the acid treatment
> that is needed to obtain BrdU staining also abolish the GFP visualization. GFP
> staining is not extremely strong to start with so we have used anti-GFP
> secondaries to improve visualization. We have manipulated several variables
> associated with the acid treatment (length, concentration, heat, pH) or
> fixation (mostly length of fixation) to no avail. This is a common problem
> with BrdU since the acid treatment interferes with some antibodies. Although
> we have used the EdU click-it system successfully, we are testing a large
> number of animals and the cost of using EdU is significant if not prohibitive.
> So, any suggestion to obtain BrdU staining while preserving endogenous GFP
> stain?
> Many thanks!
> Claude
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054

[Claude Messier]

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*****

Thank you Linda! We have been using 2N HCl but in distilled water and no triton for 1 hour.
We will try your recipe and see if it is compatible with the rest of our protocol.
Just by curiosity, which tissue are you sectioning and which fixation protocol do you use? This might introduce additional factors.
We stain mouse brains and use Lana's fixative (1 hour post-fixation) and 24 hours sucrose.

Again, thanks for the suggestion !

Claude
________________________________________
Dr. Claude Messier
School of Psychology / Ecole de psychologie
University of Ottawa / Université d'Ottawa
136 Jean-Jacques Lussier Room 2076A
Ottawa Ontario
K1N 6N5
[phone] ext 4562
FAX: [phone]

Le 2013-05-28 à 08:490, Linda Barthel <[hidden email]> a écrit :

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Claude,
> What normality is your HCl?  We have used BrdU in combination with eGFP
> with much success.  Our HCl is 2N in PBS with 0.5% triton.  Our treatment
> for cryosections is for 1/2 hr followed by 2 buffer rinses and then proceed
> with our ICC as usual.
> Regards,
> Linda Barthel
>
> 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
>
>
>
> On Tue, May 28, 2013 at 8:31 AM, Claude Messier <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Although this is not a confocal question proper, I would appreciate
>> suggestions for a immunohistochemistry problem. We are trying to visualize
>> GFP in the brain of transgenic mice expressing GFP in oligodendrocyte
>> precursor cells. In order to identify dividing oligodendrocyte precursor
>> cells,
>> we also want to use BrdU labelling. The problem is that the acid treatment
>> that is needed to obtain BrdU staining also abolish the GFP visualization.
>> GFP
>> staining is not extremely strong to start with so we have used anti-GFP
>> secondaries to improve visualization. We have manipulated several variables
>> associated with the acid treatment (length, concentration, heat, pH) or
>> fixation (mostly length of fixation) to no avail. This is a common problem
>> with BrdU since the acid treatment interferes with some antibodies.
>> Although
>> we have used the EdU click-it system successfully, we are testing a large
>> number of animals and the cost of using EdU is significant if not
>> prohibitive.
>> So, any suggestion to obtain BrdU staining while preserving endogenous GFP
>> stain?
>> Many thanks!
>> Claude
>>
>
>
>
> --
> 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

[Claude Messier]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Thank you Katrin! We'll look into that.

Best,

Claude
________________________________________
Dr. Claude Messier
School of Psychology / Ecole de psychologie
University of Ottawa / Université d'Ottawa
136 Jean-Jacques Lussier Room 2076A
Ottawa Ontario
K1N 6N5
[phone] ext 4562
FAX: [phone]

Le 2013-05-28 à 08:480, Katrin roth <[hidden email]> a écrit :

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Claude,
>
> we also used EdU click-it system. To minimalize the EdU usage we used 50ml Falcons in the drinking bottles and we generated little agarose pads with the EdU which is feeded.
>
> All the best
> Katrin
> ________________________________________
> Von: Confocal Microscopy List [[hidden email]] im Auftrag von Claude Messier [[hidden email]]
> Gesendet: Dienstag, 28. Mai 2013 14:31
> An: [hidden email]
> Betreff: Imaging GFP with BrdU
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Although this is not a confocal question proper, I would appreciate
> suggestions for a immunohistochemistry problem. We are trying to visualize
> GFP in the brain of transgenic mice expressing GFP in oligodendrocyte
> precursor cells. In order to identify dividing oligodendrocyte precursor cells,
> we also want to use BrdU labelling. The problem is that the acid treatment
> that is needed to obtain BrdU staining also abolish the GFP visualization. GFP
> staining is not extremely strong to start with so we have used anti-GFP
> secondaries to improve visualization. We have manipulated several variables
> associated with the acid treatment (length, concentration, heat, pH) or
> fixation (mostly length of fixation) to no avail. This is a common problem
> with BrdU since the acid treatment interferes with some antibodies. Although
> we have used the EdU click-it system successfully, we are testing a large
> number of animals and the cost of using EdU is significant if not prohibitive.
> So, any suggestion to obtain BrdU staining while preserving endogenous GFP
> stain?
> Many thanks!
> Claude

[Linda Barthel]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Claude,
We are looking at zebrafish retinas.  The eyecups are fixed in 4%
paraformaldehyde with 0.1M PO4 and 5% sucrose.  If Lana's contains methanol
or ethanol that will quench your GFP.
L


On Tue, May 28, 2013 at 3:14 PM, Claude Messier <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Thank you Linda! We have been using 2N HCl but in distilled water and no
> triton for 1 hour.
> We will try your recipe and see if it is compatible with the rest of our
> protocol.
> Just by curiosity, which tissue are you sectioning and which fixation
> protocol do you use? This might introduce additional factors.
> We stain mouse brains and use Lana's fixative (1 hour post-fixation) and
> 24 hours sucrose.
>
> Again, thanks for the suggestion !
>
> Claude
> ________________________________________
> Dr. Claude Messier
> School of Psychology / Ecole de psychologie
> University of Ottawa / Université d'Ottawa
> 136 Jean-Jacques Lussier Room 2076A
> Ottawa Ontario
> K1N 6N5
> [phone] ext 4562
> FAX: [phone]
>
> Le 2013-05-28 à 08:490, Linda Barthel <[hidden email]> a écrit :
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Claude,
> > What normality is your HCl?  We have used BrdU in combination with eGFP
> > with much success.  Our HCl is 2N in PBS with 0.5% triton.  Our treatment
> > for cryosections is for 1/2 hr followed by 2 buffer rinses and then
> proceed
> > with our ICC as usual.
> > Regards,
> > Linda Barthel
> >
> > 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
> >
> >
> >
> > On Tue, May 28, 2013 at 8:31 AM, Claude Messier <[hidden email]>
> wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Although this is not a confocal question proper, I would appreciate
> >> suggestions for a immunohistochemistry problem. We are trying to
> visualize
> >> GFP in the brain of transgenic mice expressing GFP in oligodendrocyte
> >> precursor cells. In order to identify dividing oligodendrocyte precursor
> >> cells,
> >> we also want to use BrdU labelling. The problem is that the acid
> treatment
> >> that is needed to obtain BrdU staining also abolish the GFP
> visualization.
> >> GFP
> >> staining is not extremely strong to start with so we have used anti-GFP
> >> secondaries to improve visualization. We have manipulated several
> variables
> >> associated with the acid treatment (length, concentration, heat, pH) or
> >> fixation (mostly length of fixation) to no avail. This is a common
> problem
> >> with BrdU since the acid treatment interferes with some antibodies.
> >> Although
> >> we have used the EdU click-it system successfully, we are testing a
> large
> >> number of animals and the cost of using EdU is significant if not
> >> prohibitive.
> >> So, any suggestion to obtain BrdU staining while preserving endogenous
> GFP
> >> stain?
> >> Many thanks!
> >> Claude
> >>
> >
> >
> >
> > --
> > 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
>

--
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