Immunostaining the Yeast Nuclear Membrane for Imaging by Super-Resolution Fluorescence Microscopy.

The ability to image biological samples beyond the diffraction limit (super-resolution) has opened a world of new discoveries in molecular biology. Super-resolution fluorescence microscopy requires specific fluorophores that often require immunostaining with labeled antibodies to achieve the necessary parameters for imaging. Budding yeast Saccharomyces cerevisiae is a common model organism used in cell biology, genetics, and molecular biology. However, yeast are difficult to immunostain due to the cell wall that hinders antibody penetration. In this chapter, I describe the methods we use for immunostaining yeast to visualize the nuclear membrane with the necessary efficiency for imaging by super-resolution fluorescence microscopy. I discuss approaches and optimizations to prepare samples for different imaging techniques including Structured Illumination Microscopy (SIM) and Stochastic Optical Reconstruction Microscopy (STORM). Super-resolution imaging can then be used to determine the precise location of proteins within the nuclear membrane, or if perinuclear proteins reside inside or outside the nucleus. The methods can be extended to other techniques requiring antibody penetration into yeast cells.