Improved light microscopic demonstration of D-amino acid oxidase activity in cryotome sections using cerium ions as capturing and amplifying agent--the Ce/Ce-H2O2-DAB procedure.
The light microscopical demonstration of D-amino acid oxidase (AAOX) activity with cerium (Ce III) as the capturing agent was improved. The incubation medium was stabilized by the employment of triethanolamine and detrane complexed cerium. A considerable increase in intensity of the reaction was accomplished by treatment of the AAOX-incubated sections with Ce III which reacted with the primary reaction product Ce IV-perhydroxide to form Ce IV-hydroxide. In this way the primary reaction product was reduced and enlarged concomitantly. The Ce IV-hydroxide was converted into Ce IV-perhydroxide by H2O2, which was visualized by blue-black stained Ni-DAB complexes. Thus, Ce III is used as capturing agent as well as amplifier (Ce/Ce-H2O2-DAB method). The primary reaction product Ce III-phosphate formed by coreacting phosphatases was selectively extracted by citrate containing glycine-NaOH buffer while Ce IV-perhydroxide remained in the sections. In model experiments it was proven that the perhydroxide groups in the Ce IV-perhydroxide compound initiate predominantly the DAB polymerization while the contribution of Ce III and Ce IV is small.
Department of Histochemistry, Friedrich Schiller University Jena, Germany.
K J
KJ
Halbhuber
H
H
Feuerstein
N
N
Zimmermann
M
M
Klinger
D
D
Kalicharan
U
U
Hupfer
D015100:3,3'-Diaminobenzidine; D000818:Animals; D002021:Buffers; D002563:Cerium; D002951:Citrates / Q000494:pharmacology; D003605:D-Amino-Acid Oxidase / Q000032:analysis*; D005260:Female; D005615:Freezing; D006652:Histological Techniques; D006861:Hydrogen Peroxide; D008297:Male; D008853:Microscopy / Q000379:methods*; D010084:Oxidation-Reduction; D051381:Rats; D011919:Rats, Inbred Strains; D016707:Tissue Fixation
D002021:Buffers; D002951:Citrates; D015100:3,3'-Diaminobenzidine; D002563:Cerium; D006861:Hydrogen Peroxide; D003605:D-Amino-Acid Oxidase