talulahgosh <@t> gmail.com
Tue Nov 18 11:09:13 CST 2008
In response to the email below:
We use a separate secondary label with our GFP antibody and it doesn't work
for us, so that's probably not the problem.
Our secondary is from Jackson Immuno, Cy2.
Also, we do antigen retrieval with a microwave, which also doesn't help.
On Tue, Nov 18, 2008 at 12:03 PM, Gayle Callis <gayle.callis <@t> bresnan.net>wrote:
> The problem may be that you are using a directly conjugated antiGFP rather
> than a rabbit antiGFP followed by coming back with a secondary conjugated to
> Alea Fluor 488. This is called quenching, a phenomenom, where fluorophore
> moleculas too close together on cells or in tissues tend to cancel out the
> fluorecsing ability of the fluorophore. You should go onto internet and
> look at a fluorescence Jablonski diagram which show how this occurs, Olympus
> website also wonderful discussions in pdf form, for all fluorescence
> applications, including this diagram - for confocal and fluorescent
> I suggest you stain for GFP (Teri Johnson method) where you retrieve, use a
> rabbit antiGFP, then come back with a secondary either conjugated to FITC
> (Jackson has excellent antibodies, or one of the Cy fluorophores, or better
> yet, Goat antirabbit-Alexa 488. Be sure you use Molecular Probes Prolong
> gold antifade mounting media after staining - this is superior for
> preventing fading of fluorophores, even 488. Not all aqueous mounting
> medias will prevent fading of fluorophores, even the Alexa dyes.
> Teri recommends rabbit antiGFP rather than Goat antiGFP for paraffin work,
> as the rabbit hosted antibody gave less background than the goat antiGFP.
> One can also purchase Rabbit antiGFP from Rockland.
> I made a CC to Teri Johnson so she is in this email loop. You may want to
> discuss this problem with her, and what antigen recovery method she prefers.
> If worse comes to worse, and you can't afford another antibody, use the
> antiGFP-488, come back with an antiAlexa 488 (Molecular Probes) and detect
> that antibody with an antibody that has FITC, or the appropriate
> fluorophore. A round about way, but the same type of technic used to detect
> Gayle M. Callis
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