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One possible check on this if you have a spectral scanning module is to look
at spectra. Muscle cell autofluorescence is distinguishable from most probe
signals by its spectral signature. I have done this effectively on the
Zeiss LSM510 Meta.
D. Scott Snyder, Ph.D.
Lab Manager, Integrated Microscopy Core
Baylor College of Medicine
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Louis Villeneuve
Sent: Wednesday, January 21, 2004 9:20 AM
To: [log in to unmask]
Subject: Re: Cardiomyocytes
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Hi Fredrik,
I also get this kind of staining in mouse, and dog heart ventricle cells. In
the Fitc range, I think that can be autofluorescence. there was a subject
on this list 1 or 2 years ago. I will take a look in the archives...
Also, i have seen these kind of pattern with other wavelenths (triTC, Cy5)
and the negative controls were looks good. For these experiments, I was
blocking the isolated cells with 10%serum. I have reduced my blocking
buffer concentration (to +/- 2% serum) and the recurrency of this staining
had dissapeared. does too much blocking can cause non specific stainng?
This is an option that I leave open...
Best regards
Louis
Louis R. Villeneuve
Assistant de recherche -microscope confocal
Institut de Cardiologie de Montréal- Centre de Recherche
5000 est Bélanger, Montréal
Québec , Canada
H1T 1C8
514-376-3330 ext3511
514-376-1355 (Fax)
----- Original Message -----
From: "Fredrik Swift" <[log in to unmask]>
To: <[log in to unmask]>
Sent: Wednesday, January 21, 2004 5:15 AM
Subject: Cardiomyocytes
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hello,
>
> I'm labelling rat cardiomyocytes with different antibodies. Sometimes
> I
get
> what I think is unspecific staining. The staining consists of
> longitudinal "strips" with low fluorescence intensity (Alexa-488
> labelled 2nd ab's).
This
> is typical if I use very low concentrations of primary ab's, and I get
> similar results in negative control exp. with no primary ab.
> Apparently,
the
> 2ary ab is the cause of this, but I cannot explain what it stains. Is
> it myosin/actin that I see? Have anyone had the same results
> previously? Any help would be appreciated! I can send jpg.-files of
> the images if the description wasn't good enough.
>
> Regards,
> Fredrik
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