Single antibody procedure incubation time

To double label tissue with an antibody and phalloidin, follow the standard procedure for antibody labeling (see Protocol 12.2 [Labeling with a Single Antibody]) and use a fluorescent secondary antibody (step 6). Following the secondary antibody incubation, wash tissue three times in PBS/TX (10 minutes each wash). Incubate in phalloidin, rinse in PBS, and mount as outlined in steps 3-6 above. 

BrdU/DNA flow cytometry offers flexibility and diversity in the study of cell kinetics from cells in culture to human tumors in vivo. The essence of the procedure is to pulse label with BrdU by a short-term incubation in vitro or by a single injection in vivo samples are then taken at time intervals thereafter and stained after fixation in ethanol. The cells are then stained with a monoclonal antibody against BrdU that can be either directly conjugated to a fluoro-chrome (usually fluorescein isothiocyanate [FITC]) or, alternatively, bound to a second antibody conjugated with FITC. The cells are then counterstained with propidium iodide (PI) to measure the DNA content and analyzed on the flow cytometer. The results are displayed as linear-red fluorescence on the x-axis vs linear or log-green fluorescence on they-axis. 

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