High‐pressure freezing followed by freeze substitution of a complex and variable density miniorgan: the wool follicle

Abstract

Cryofixation by high-pressure freezing (HPF) followed by freeze substitution (FS) is a preferred method to prepare biological specimens for ultrastructural studies. It has been shown to achieve uniform vitrification and ultrastructure preservation of complex structures in different cell types. One limitation of HPF is the small sample volume of <200 µm thickness and about 2000 µm across. A wool follicle is a rare intact organ in a single sample about 200 µm thick. Within each follicle, specialized cells derived from multiple cell lineages assemble, mature and cornify to make a wool fibre, which contains 95% keratin and associated proteins. In addition to their complex structure, large density changes occur during wool fibre development. Limited water movement and accessibility of fixatives are some issues that negatively affect the preservation of the follicle ultrastructure via conventional chemical processing. Here, we show that HPF-FS of wool follicles can yield highquality tissue preservation for ultrastructural studies using transmission electron microscopy.

Processing hair follicles for transmission electron microscopy

“…94 An approach that combines acetone and hexylene glycol has been effective for dehydrating wool follicles. 25,68 Hexylene glycol is a class of alcohol that acts as an emulsifier. Since it is miscible with both water and lipid molecules, it can solubilize sebaceous and adipocyte lipids that potentially act as a barrier to dehydration.…”

“…A hybrid fixation approach, in which follicles are cryogenically fixed using a high-pressure freezer and then prepared for ambient temperature microscopy through freeze substitution can be used for ultrastructural and immunohistochemical TEM. 25,72 Sample preparation for different imaging endpoints is achieved by varying the freeze-substitution protocol. For ultrastructural studies, a cold (−90°C) cocktail of acetone, glutaraldehyde and OsO 4 is used.…”

“…For instance, interaction between acetone and uranyl acetate results in uranium salt precipitation. 25,92,100 One advantage of freeze substitution is that solvent-fixative or fixativefixative interactions do not occur at low temperatures. 44,101 Acetone and methanol are widely preferred as solvents for freeze substitution.…”

“…6,50,81,102,103,126 However, the additional level of ultrastructural detail acquired following post-staining with uranyl acetate and lead citrate was remarkable. 25,34,68,127 For immunoelectron microscopy, follicular sections were first labelled with primary and secondary antibodies. 62,113,128 If silver enhancement was required, grids were poststained immediately following this procedure.…”

“…The development of electron tomography and volume microscopy has aided in the 3D visualization of the precise geometry of complex macromolecular, subcellular or cellular structures including interaction of keratins and desmosomes. 25,[130][131][132] CLEM of high-pressure frozen and freezesubstituted samples, rather than a conventionally processed sample, can be ideal for simultaneously preserving the ultrastructural and antigenic epitopes; and dark-field scanning transmission electron microscopy (DF-STEM) then enables visualization of immunogold markers at high-resolution without the need for any post-staining or silver enhancement procedures. 71 While advancements in electron tomography have enabled the 3D visualization of macromolecular process, a limited sampling volume limits the investigation of features that span more than a few…”