Purpose: To investigate the histochemistry of the tear film, conjunctival goblet cells and corneal superficial epithelial cells by an in vivo cryofixation with the freeze-substitution method.
Methods: Following rapid freezing of the eyeballs by an isopentane-propane mixture cooled by liquid nitrogen directly, the specimens were freeze-substituted and then embedded in Lowicryl K4M resin at low temperature, or in epoxy resin for histochemical study, using light and transmission electron microscopy.
Results: The tear film was weakly positive with periodic acid-Schiff staining, but negative with alcian blue at pH 2.5 or pH 1.0 and high iron diamine staining. Conjunctival goblet cells were positively stained by all these techniques. There was a gradually weakening staining intensity of mucus from the goblet cells to the conjunctival surface. With the periodic acid-thiocarbohydrazide-silver protein method the tear film was weakly stained, while glycocalices, vesicles, glycogen in epithelial cytoplasm and the mucus secretory granules of goblet cells were all intensely stained.
Conclusions: The tear film consists mainly of dilute mucus and contains either neutral or acid glycoproteins.