Histochemical study on rat tear film and ocular surface epithelial cells

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 him 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.