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 lei...@buffalo.eduwrote:
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
lei...@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.pa...@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
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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
lei...@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.
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http
