Cytochrome oxidase histochemistry in the rat hippocampus. A quantitative methodological study.
The diaminobenzidine (DAB) method was adapted for the microphotometric determination of cytochrome c oxidase (cyt ox) in the rat hippocampus. The qualitative and quantitative investigations at the light microscopic level showed that acetone and cytochrome c pretreatment of cryostat sections resulted in a significant increase of demonstrable cyt ox activities. The final incubation medium consisted of 7.5 mM DAB, 2% polyvinylalcohol (PVA) and 6% dimethyl sulfoxide in 0.1 M Hepes buffer; final pH 7.5. PVA was used to keep DAB and artificially oxidized DAB in solution. In the kinetic and endpoint measurements a linear response of the reaction with highest slope was observed only in the initial 5-6 min of reaction. Thereafter the slope decreased. Ultracytochemical demonstrations, which were performed as a topochemical control, showed reaction product only in mitochondria (cristae and intermembranous space). In contrast to vibratome sections all mitochondria reacted positively in cryostat sections of aldehyde-fixed hippocampi. The enhancement of reaction after acetone pretreatment of cryostat sections (light microscopic level) and after a freezing step in ultracytochemistry is discussed in connection with diffusion problems of DAB through mitochondrial membranes.
Kugler
P
P
Department of Anatomy, University of Würzburg, Federal Republic of Germany.
Vogel
S
S
Volk
H
H
Schiebler
T H
TH
D015100:3,3'-Diaminobenzidine; D000818:Animals; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006624:Hippocampus / Q000378:metabolism* / Q000648:ultrastructure; D006651:Histocytochemistry; D008297:Male; D008854:Microscopy, Electron; D008928:Mitochondria / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D015100:3,3'-Diaminobenzidine; D003576:Electron Transport Complex IV