[Histonet] Problem wih FITC secondary "unbinding" after about 10 hours (using immunofluorescence)

[Merced M Leiker]

We use a range of Alexa Fluors (including 488) all the time with either 
SlowFade Gold or ProLong Gold antifade mounting media on every primary 
antibody we use. We can still see the label a week or more later, 
especially if we store the slides at -20 C.

Alexa Fluors are very bright and stable compared to traditional 
fluorophores like FITC, TRITC...

Another consideration is are you doing confocal or epifluorescent imaging? 
Confocal will bleach your sample sooner.

The Alexa Fluors and mounting media mentioned above are all available from 
Invitrogen at the following links (this is NOT a plug for Invitrogen, 
though it may appear that way; we are an independent lab!):

<http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/alexa-fluor.html>
<http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/SlowFade-Gold.html>
<http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/ProLong-Antifades-Brand-Page.html>

Regards,

Merced M Leiker
Research Technician II
Cardiovascular Medicine
348 Biomedical Research Building
State University of New York at Buffalo
3435 Main St, Buffalo, NY 14214  USA
leiker <@t> buffalo.edu
716-829-6118 (Ph)
716-829-2665 (Fx)

No trees were harmed in the sending of this email.
However, many electrons were severely inconvenienced.

--On Tuesday, October 27, 2009 10:36 AM -0700 Janelle Pakan 
<janelle.pakan <@t> gmail.com> wrote:

> We are having a very strange problem with our FITC-conjugated secondary in
> double labeling immunofluorescence processing. We are processing for 3
> different enzymes using a Rhodamine secondary and these seem to be fine
> throughout the process, but using 3 different markers (GFAP, MAP2, OX42)
> with a FITC conjugated secondary we see something very strange. The
> labeling looks really nice immediately after processing and mounting for
> up to 6-10 hours later, then when left in the fridge overnight, the next
> day the FITC (and only the FITC, the rhodamine is fine) seems to "unbind"
> and is all over the tissue creating a "background" type fluorescence.
>
> I have tried 4 different secondaries from 3 different companies (although
> they are all FITC conjugated - want to try a cy2 or alexafluor 488 but
> don't have any yet) and they all do they same thing - really nice
> immediately after processing, but cell labeling is "gone" by the next day
> and appears as specks all over the tissue sample.
>
> We are using 4%PFA fixed, floating rat brain sections (used three
> different brains so far and all had the same effect).
>
> These are the other things I checked:
> - used 3 different mounting media (Fluoromount-G, Elvanol, 90% glycerol):
> all had same effect. Leaving tissue overnight in PBS in fridge also had
> same effect, so figure it cant be anything with the mounting process. -
> used 3 different primaries and all have same effect
> - we use ovoid (BR0014G) PBS tablets (1 per 100ml, pH 7.3) - commercially
> available standard PBS tablets... should be okay?? Anyone else use this?
>
> Any help would be appreciated, or just to know if anyone has seen this
> before. I did immuno for 5 years in a different lab and never saw anything
> like this, but recently switched labs and had this problem immediately. I
> figure it must be something in the solutions I am using n the new lab,
> but I just cant fathom what it would be at this point.
>
> Thanks
>
> Dr. Janelle Pakan
> University of British Columbia
> Brain Research Centre
> 2211 Westbrook Mall
> Vancouver, BC
> Canada
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Merced M Leiker
Research Technician II
Cardiovascular Medicine
348 Biomedical Research Building
State University of New York at Buffalo
3435 Main St, Buffalo, NY 14214  USA
leiker <@t> buffalo.edu
716-829-6118 (Ph)
716-829-2665 (Fx)

No trees were harmed in the sending of this email.
However, many electrons were severely inconvenienced.