PAIS at wp.path.uab.edu wrote:
>To better understand this problem, it would be necessary to know what
>kind of background (what specific types of tissue microenvironments show
>background). A prblem with Fc mediated background as suggested by the
>last posting has a characteristic appearance due to the distribution of
>cells with high FcR levels.
My cells which I am interested in analysing are splenocytes. The
postivity can vary, as the background is concentration dependent.
The infectious virus is there but I can not see the antigen due to the
background, at any dilution. As mentioned prior, I have blocked with
an Fc block and with normal goat serum to no avail.
>When you say the "CD antigens" have worked well, exactly what do you
>mean? Do you use the same secondary reagent for these as for the
>murine mAb for the virus?
The CD antigens are CD4,CD8 and B220 (6B2 clone). All are rat MAb's
that are directly conjugated to R-PE. I am double labelling these
cells and do not get cross reactivity with the rat, as the background
with the anti-virus Ab alone is the same. They work well in that
they are always very clean with at least a log of fluorescent
separation of positive cells, and the % of CD4, CD8 and B-cells is
very consistent.
Hope this helps clarify the above questions!
Dan
