Have you looked at a section with no antibody to see if there is simply
an autofluorescence problem? Autofluorescence of red cells and collagen
is very common. Depending on the filter/wavelength you're using you can
quench some autoflourescence by using a chromagen counterstain such as
trypan blue. Another way to determine autofluorescence is to switch
between filters (i.e. FITC to TRITC). Cells will continue to
autofluoresce between filters whereas true chromagen fluorescence will
only fluoresce in the appropriate filter. The exception to this being
that you can get some "burn through" to other filters by using extremely
large amounts of fluorochrome (i.e.via tyramide over-amplification).
Lastly you're already using a maximal conc. of blocking agent. Its my
opinion that you cannot block your way out of nonspecific background
problems. You should begin by diluting the primary antibody perhaps in
conjunction with using a signal amplification system (i.e. tyramide).
sean troth
Dr. Douglas Darling wrote:
>I am using an FITC-conjugated secondary antibody on histological
>sections of mouse embryos. We get great labeling with our primary
>antiserum. However, all the blood cells also label. The blood cells
>fluoresce brightly even when no primary antibody is used. I use 10%
>serum-0.1% triton to block. There is obviously some non-specific
>binding of the secondary Ab-FITC to the blood cells. I tried 4
>different secondary Ab conjugated to either FITC or TRITC. HOW CAN I
>BLOCK THIS???
>>---
>
