Improved preservation of alkaline phosphatase in salivary glands of the cat

Synopsis

The effects of tissue preparation on the localization of alkaline phosphatase were assessed at the light and electron microscopical levels. Glutaraldehyde-containing solutions were more inhibitory than formaldehyde alone but appeared to give betterin situ immobilization of the enzyme. The inhibitory effects of glutaraldehyde solutions were related especially to temperature and time and not necessarily to material absorbing at 235 nm. Distilled glutaraldehyde was the only form of glutaraldehyde to give consistently good results with least inhibition. Snap-freezing of pre-fixed tissue, after washing in a sucrose-containing solution, gave satisfactory results without undue ice-crystal formation. Immersion in dimethylsulphoxide before snap-freezing gave less good localization with greater diffusion of reaction product. Use of a Sorvall tissue-chopper on unfrozen tissue did not produce satisfactory sections. Free-floating sections of pre-fixed material appeared less inhibited than glass-mounted sections. The most satisfactory results were obtained after per-arterial perfusion with a 1% distilled glutaraldehyde-0.8% formaldehyde mixture, containing dextran, for up to 5 min followed by formaldehyde-dextran. The results were uniform; there was stronger staining of stromal surfaces of myoepithelial cells than previously, basal duct cells were also stained and occasional staining between acinar cells was now evident for the first time.