The cerium perhydroxide-diaminobenzidine (Ce-H2O2-DAB) procedure

Abstract

New light microscopic visualization methods were developed for the histochemical detection of non-specific alkaline and acid phosphatase, Mg-, Ca- and Na, K-dependent adenosine triphosphatase, myosin adenosine triphosphatase, glucose-6-phosphatase, 5'-nucleotidase and thiamine pyrophosphatase with cerium ions as trapping agents in cryostat and plastic sections. The techniques are based on the conversion of cerium phosphate into cerium perhydroxide by H2O2 which decomposes at 55 degrees-60 degrees C into cerium hydroxide and oxygen radicals. These radicals are able to oxidize diaminobenzidine (DAB) to DAB brown. Addition of nickel ions to the DAB-H2O2 mixture generates bluish-black stained nickel-DAB complexes. Compared with the classical metal precipitation, azo, azoindoxyl and tetrazolium procedures the H2O2-DAB and especially the H2O2-DAB-nickel methods provided identical or superior results in catalytic phosphatase histochemistry and immunohistochemistry when using non-specific alkaline phosphatase as the enzyme label.

Angiotensin II AT1 receptors and Na+/K+ ATPase in human umbilical vein endothelial cells

“…Briefly, cells were incubated for 45 min at 37 C in a medium containing 2 m p-nitrophenyl phosphate, 2 m CeCl 3 , Mg 2+ and K + as activators and the non-specific alkaline phosphatase inhibitors, levamisole and -phenylalanine, in 50 m Tricine buffer (Sigma), pH 7·5. Then the cells were treated for 15 min at 60 C using the visualization medium of Halbhuber et al (1988) containing 1·4 m 3,3 -diaminobenzidine, 100 m NaN 3 and 12 m Ni(NH 3 ) 6 SO 4 in Tris-HCl, and 0·002% H 2 O 2 . Control cells were incubated without substrate, with 10 m ouabain (Sigma), or in medium in which potassium ions were replaced by equimolar concentrations of sodium ions.…”

Immunoalkaline phosphatase with cerium‐based cytochemistry for double staining at the transmission electron microscopic level

“…One disadvantage of the cerium method is that the reaction product is not visible at the light microscopic (LM) level. Various manipulations involving either sulphide conversion or the production of perhydroxide groups and subsequent deposition of visible diamino-benzidine reaction production have been published (Halbhuber et al, , 1988Van Goor et al, 1989). These methods were not investigated herein.…”

Corticosteroid induction of renal and intestinal K+-dependentp-nitrophenylphosphatase in young and adult rats

“…The specimens of distal colon were incubated for 75 rain to accumulate more reaction product, due to low enzyme activity. The rinsed sections were treated for 15 rain with the visualization medium of Halbhuber et al (1988) at 55-60~…”