I tried incubating the mouse tissue with an unconjugated IgG (for 2 hours) prior to adding the mouse secondary, but I still get a lot of background. Is there a way to reduce the background without using a MOM kit?
Jane Lindborg
These are frozen fixed tissue sections, 10 um. Rehydrate with PBS, block in 10% NDS for 1h, primary antibody overnight at 4C, wash with PBS (3X30 min), incubate in unconjugated affinity purified F(ab) fragment anti-mouse IgG (H+L) for 2 h, followed by donkey anti-mouse secondary for 1h at RT, wash PBS 3x30 min.
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Sven Reister
It is always unfavorable to use mouse Ab for mouse tissue because of residential immune cells. The easies way would be to switch the host species of primary Ab or to take a direct conjugated Ab
Regards
Sven
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