Freeze‐substitution without aldehyde or osmium fixatives: ultrastructure and implications for immunocytochemistry

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Abstract
SUMMARY: Cryo‐fixation followed by freeze‐substitution without aldehyde or osmium fixation has been investigated as a method for preparing biological specimens with a view to minimizing antigenic alteration. Samples of both solid tissues (mouse small intestine and human kidney) and a human tumour cell line grown in vitro were rapidly frozen by impact (slammed) onto a copper block cooled with liquid nitrogen. They were freeze‐substituted at −80°C in methanol, and embedded at low temperature in Lowicryl K4M or HM20. Resin blocks were polymerized by ultraviolet light. Well‐preserved ultrastructure was observed in the outer 10–15 μm of all samples. Positive immunocytochemical localization of fixation‐resistant and fixation‐labile antigens was obtained on sections of human kidney and the human breast tumour cell line ZR‐75‐1 at both light and electron microscope levels.