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High‐pressure freezing followed by freeze substitution of a complex and variable density miniorgan: the wool follicle
Author(s) -
VELAMOOR S.,
RICHENA M.,
MITCHELL A.,
LEQUEUX S.,
BOSTINA M.,
HARLAND D.
Publication year - 2020
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/jmi.12875
Subject(s) - ultrastructure , cryofixation , wool , follicle , keratin , transmission electron microscopy , biophysics , chemistry , tunica albuginea (penis) , hair follicle , vitrification , anatomy , biology , microbiology and biotechnology , materials science , botany , composite material , nanotechnology , andrology , medicine , paleontology , penis
Summary 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 high‐quality tissue preservation for ultrastructural studies using transmission electron microscopy.