Samples needed for training of standard immunofluorescence microscopy.
Stained sample(s).
Secondary control(s).

In addition to doing the experiment robustly for your own knowledge, many reviewers will ask to see the secondary only controls.

For each channel, the exposure should be set with the brightest sample for each channel. There should be no saturation. Most imaging systems have dynamic range wider than may be displayed linearly on the monitor. Signal in the darks may be shown by adjusting the LUT, not by saturating the raw data intensities.

On the confocal, the offset should always be set at zero. If there is signal in the secondary control, this may be adjusted later digitally or should be addressed in tissue preparation by bleaching (e.g. sodium periodate, glycine) or blocking. To addess bleaching vs blocking as solution to the problem, tissue treated with all steps except addition of any fluorescent probes is a good control.

Images of the experimental samples and the secondary control (and other controls) should be imaged at the same settings.

Why not turn down the offset? Because it may throw away real data. There may be specific real labeling of sparse molecules that have their intensity masked by autofluorescence or non-specific binding. By reducing the background in the tissue preparation, contrast will be increased and real weak signal may be visible. This is critical to properly address scientific inquiries.

Live cell concerns (rough draft)

If doing live cell imaging with additions of dyes (such as mitotracker) before doing the full experiment, tests should be done to use minimum staining required and instructions should be followed as additions of probes may impact the biology. For instance, behavior of cells, phenotype should be the same before & after expressions of FPs.