Problems with a fluorescently labelled antibody.

[Shane van Breda]

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Dear members,

I am having problems with my fluorescently labelled antibody (conjugate is
FTC-ED: ex: 488nm and em: 525nm).

Can anyone give me advice on how to test my antibodies flourescence
capabilities on the confocal? I just would like to know that they can actually
fluoresce.

I would like to view its fluorescence without staining samples if possible.

Thank you very much.

Shane Vontelin van Breda.

P.S: Does anyone have experience with freeze drying samples and resin
infiltration?

[Gary G. Li-2]

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Hello Shane Vontelin van Breda,


*** Testing fluorescently labeled antibody ***


If you have an inverted microscope, you can use a dish with glass coverslip,
and place a drop of the solution of the fluorescently labeled antibody on
the coverslip.  Both an inverted widefield fluorescent microscope and a
confocal microscope will work.


If you use an upright fluorescent microscope, you can simply place a drop of
the solution onto a slide and cover it with a coverslip.


Gary G. Li, Ph.D.

Johns Hopkins School of Medicine.

Baltimore, MD 21224.


On Mon, Dec 6, 2010 at 2:53 AM, Shane van Breda <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear members,
>
> I am having problems with my fluorescently labelled antibody (conjugate is
> FTC-ED: ex: 488nm and em: 525nm).
>
> Can anyone give me advice on how to test my antibodies flourescence
> capabilities on the confocal? I just would like to know that they can
> actually
> fluoresce.
>
> I would like to view its fluorescence without staining samples if possible.
>
> Thank you very much.
>
> Shane Vontelin van Breda.
>
> P.S: Does anyone have experience with freeze drying samples and resin
> infiltration?
>

[Martin Wessendorf-2]

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On 12/6/2010 8:17 AM, Gary G. Li wrote:

> If you have an inverted microscope, you can use a dish with glass coverslip,
> and place a drop of the solution of the fluorescently labeled antibody on
> the coverslip.  Both an inverted widefield fluorescent microscope and a
> confocal microscope will work.
>
> If you use an upright fluorescent microscope, you can simply place a drop of
> the solution onto a slide and cover it with a coverslip.

In addition, if you have a hand-held UV light (e.g., for examining
gels), just darken the room and hold the antibody vial up to the UV
light.  The solution should fluoresce green.

Martin Wessendorf
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Craig Brideau]

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Some dyes don't fluoresce unless they are bound to something.  Could that be
the case here?  I don't know because I don't use that one myself, but I'm
sure someone on the list does...

Craig



On Mon, Dec 6, 2010 at 7:36 AM, Martin Wessendorf <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> On 12/6/2010 8:17 AM, Gary G. Li wrote:
>
>  If you have an inverted microscope, you can use a dish with glass
>> coverslip,
>> and place a drop of the solution of the fluorescently labeled antibody on
>> the coverslip.  Both an inverted widefield fluorescent microscope and a
>> confocal microscope will work.
>>
>> If you use an upright fluorescent microscope, you can simply place a drop
>> of
>> the solution onto a slide and cover it with a coverslip.
>>
>
> In addition, if you have a hand-held UV light (e.g., for examining gels),
> just darken the room and hold the antibody vial up to the UV light.  The
> solution should fluoresce green.
>
> Martin Wessendorf
> --
> Martin Wessendorf, Ph.D.                   office: [phone]
> Assoc Prof, Dept Neuroscience                 lab: [phone]
> University of Minnesota             Preferred FAX: [phone]
> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
> Minneapolis, MN  55455                    e-mail: [hidden email]
>

[Kate Luby-Phelps]

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The fluorescence of FITC will be strongly dependent on pH. You might try pH 8.0
to see if it helps. Also, since FITC bleaches very fast, if the solution is dilute, it
is possible (although unlikely) that it has faded already by the time you get a
good look, especially with confocal. Alexa488 would be a better choice.