Help! larval brains & Texas Red

Chris Jones jonesc at cshl.org
Wed Oct 25 12:55:09 EST 1995


I'm trying (and trying and trying) to get BrdU staining of larval brains
working in our lab. After several tries with a variety of
antibody-fluorochrome combinations, I'm still stuck. What we've decided to
use is:

a Mouse anti-BrdU monoclonal primary

a Goat anti-Mouse secondary conjugated to biotin

avidin conjugated to Texas Red

The current problem is that our negative control (brains which have not
been exposed to BrdU) show tremendous signal, glowing very brightly
(albeit uniformly). We pre-block with the appropriate normal serum before
each antibody incubation step and include the same serum with each
antibody. BSA is present throughout all steps, including washes, which are
numerous (6 x 30 min at rm temp). The brains are washed after incubation
with each of the three reagents above.

So, does anyone have any suggestions as to what we should be doing in
addition to reduce our background? Any help would be greatly appreciated.

-- 
Chris Jones (jonesc at cshl.org)



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