Silvia wrote:
Does anyone out there have experience with non-specific nuclear
staining artifact?
I've been having problem with immunostaining smooth muscle alpha
actin (Sigma A-2547) on cultured human smooth muscle cells.
I did a few optimizing test on the antibody. It was working fine at
1:400, stained the cytoplasm nicely and nice & blue counter stained
nuclei with haematoxylin.  The antibody's been frozen in aliquotes.
Three four months later I use it again, it stains all nuclei dark brown. All
nuclei.
Any suggestion?
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Dear Silvia:
Clustering of your primary antibody seems to me the most obvious reason for
your background problem (I presume your negative control is oke). Possible
remedies are:
- (ultra)centrifugation of the diluted antibody. Normally I use  an
eppendorf centrifuge at max speed for 10 min.
- increase of pH of the antibody solution to 8-9.
- addition of sodium chloride (0.5-1.5M).
To prevent clustering of future batches: add glycerol in a 1:1 ratio before
freezing.
Hope this is of help.
Peter


========================================
Peter van de Plas
AURION
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