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

[Janelle Pakan]

Thanks Merced,

We are doing confocal, but the problem doesnt seem to be bleaching - the
fluorescence is still there, it is just no longer concentrated within the
cells we are staining for, but rather dispersed all over the tissue. Almost
like it "leaked" out of the cells we were staining.

As I mentioned, I dont think it can be the mounting media, as when we left
the tissue in PBS overnight without mounting it, the signal was still gone
the next day.

Must be some sort of solution problem... maybe salts, pH, fixative?? But we
are using standard protocols and comercially available PBS. I am stumped...

Dr. Janelle Pakan
University of British Columbia
Brain Research Centre
2211 Westbrook Mall
Vancouver, BC
Canada



On Tue, Oct 27, 2009 at 11:29 AM, Merced M Leiker <leiker <@t> buffalo.edu>wrote:

> 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.
>
>