Electron microscopical demonstration of alkaline phosphatase activity with the cerium-based method in citrate-containing medium at pH 9.3 and the influence of glutaraldehyde fixation.
A cerium-based incubation medium, developed for the light microscopical demonstration of alkaline phosphatase activity, was tried out for the electron microscopical demonstration of this enzyme in kidney and heart muscle of the rat. The medium is very stable and the pH is in the optimum range of the enzyme. The medium consists of 14 mM CeCl3, 11 mM Na-citrate, 4 mM MgCl2, 10 mM p-nitrophenyl phosphate, 0.18 M glycine/NaOH buffer, pH 9.3. Other concentrations of cerium and citrate were tried out as well but 14 mM CeCl3, and 11 mM Na-citrate gave the best results with a small amount of non-specific reaction product in the nucleus that can be largely avoided by postincubation rinsing in cerium-containing buffer. In the kidney reaction product was only present along the microvilli of the proximal tubular epithelial cells. In the glomerulus no reaction product could be found whereas light microscopical cryotome sections contained activity in the glomerulus. Replacement of glutaraldehyde by formaldehyde fixatives resulted in reaction product in glomerular and tubular basement membranes, on podocyte plasma membranes and in tubular basal infoldings. In glutaraldehyde-fixed heart muscle, reaction product was present in the basement membranes and on lateral plasma membranes of endothelial cells of blood capillaries.
Hulstaert
C E
CE
Department of Histology and Cell Biology, University of Groningen, The Netherlands.
Halbhuber
K J
KJ
Kalicharan
D
D
D000469:Alkaline Phosphatase / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D002563:Cerium; D002951:Citrates; D004848:Epithelium / Q000201:enzymology / Q000648:ultrastructure; D005976:Glutaral; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007678:Kidney Glomerulus / Q000201:enzymology* / Q000648:ultrastructure; D007687:Kidney Tubules, Proximal / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D008854:Microscopy, Electron / Q000379:methods; D009206:Myocardium / Q000201:enzymology* / Q000648:ultrastructure; D051381:Rats; D017208:Rats, Wistar
D002951:Citrates; D007202:Indicators and Reagents; D002563:Cerium; D000469:Alkaline Phosphatase; D005976:Glutaral