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An alkaline phosphatase-coupled anti-Dig Fab-fragment (Roche Diagnostics) applied in blocking solution was allowed to bind to the labeled probe for two hours.
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The blocking solution was removed and 40 μl of rat anti-HA antibody (Roche; 1 100) were allowed to react 90 min at room temperature or overnight at 4°C in a wet chamber.
Biotin blocking solution was added, samples were incubated and centrifuged.
Ultrablock (blocking solution) was from Serotec (Raleigh, NC).
The excess blocking solution was removed with filter paper.
Primary antibody (in blocking solution) was incubated for 60 min and then washed three times with blocking solution.
The blocking solution was replaced with fresh solution containing primary antibodies.
Ionic blocking solutions were used to isolate capacitive currents.
Blocking solution was removed and primary antibody in blocking solution applied at the required dilution.
Blocking solution was used to block the endogenous peroxidase activity.
For membrane blocking, a 5% Top-Block solution was used.
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