To evaluate a specific cell membrane epitope, primary and secondary antibody concentrations must be optimized to minimize background signals. Briefly, to achieve an optimal image absent nonspecific signal, perform preliminary experiments using combinations of primary and secondary antibody concentrations on negative control preparations (cells not expressing the epitope). Select as upper limits the primary and secondary antibody concentrations that yield no signal. Then, use these concentrations for the IGL method on cell samples.

Coverslips for light microscopy can be used as a cell growth substrate. It is important to seed the cells a few days before the experiment (the timing varies depending on cell types used) in order to obtain 70–80% semi-confluence. If the cells require a specific substrate for growth (i.e., collagen, fibronectin, etc.), cover the glass slides with the appropriate substrate prior to seeding the cells.

Grid square area can be selected as a function of cell surface morphology and marking intensity. For example, if the cell surface is rough and/or presents a high degree of marking, it is more appropriate to select a grid with a reduced square area (3 or 5 μm²). In contrast, if the cell surface is flat and/or presents a low degree of marking, select an enhanced surface square area (15 or 20 μm²).