A correlative fluorescent and electron microscopic technique for ultralocalization of trichocyte keratins

Trichocyte keratin heterodimers in the cortex of hair occur as intermediate filaments (IF) within geometrically organized bundles (macrofibrils). To understand how heterodimerization of various keratin species contributes to IF and macrofibril formation, it is important to track the sub-cellular relationship between different keratin types in hair follicles. Distribution and localization of keratins, at an ultrastructural level, can be determined through immuno-ultrastructural studies. However, the conventional fixative-tissue interaction, dehydration and plastic embedding process, along with heavy metal staining potentially obscures the antigenic epitopes accessibility. Here, we demonstrate a sample preparation method for confocal fluorescent and electron microscopy of high-pressure frozen and freeze-substituted wool follicles that provides clear ultrastructural preservation and keratin labelling while minimizing common artefacts associated with conventional fixation. The contrast obtained using dark-field scanning transmission electron microscopy (DF-STEM) is sufficient to obtain high-resolution images without any further post-staining or silver enhancement.