Cytochrome oxidase histochemistry in the rat hippocampus

Abstract

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.

Quantitative analysis of vascularization and cytochrome oxidase following fetal transplantation in the contused rat spinal cord

“…Until use they were stored at -70°C and subsequently reacted with diaminobenzidine (DAB). Such an approach was used in order to reduce variability in reaction rates induced by tissue fixation and to allow the short incubation times necessary to insure the linear progression of the DAB reduction process (van Raamsdonk et al, 1987;Kugler et al, 1988). All experimental and control sections for quantitation were reacted at the same time to avoid artifactual staining intensity differences that could evolve from slight variations in protocol.…”

An ultracytochemical study of cytochrome oxidase activity in rat metaphyseal bone cells.

“…This was done to avoid variable fixation effects which might cause heterogenous staining patterns of the enzyme (10) employed prolonged incubation times to obtain sufficient reaction product. We incubated the specimen for 120 min or 180 min according to the previous reports (10,13,23) and found no further change at the longer incubation time. Moreover, some frozen specimens were also employed in the present study in order to facilitate penetration of DAB and cytochrome c into mitochondria during incubation according to Kugler et al (13).…”

“…Heterogenous staining patterns of cytochrome oxidase activity have also been reported among mitochondria of liver (11), spinal neurons (26), alveolar epithelial cells in lung (9, 10), chondrocytes (28) and osteoclasts (21). Several reports indicate that cytochemical reaction of cytochrome oxidase is affected by composition of the fixative (3), fixation time (23), incubation time (23) and permeability of DAB and cytochrome c (13). To minimize the inhibitory effects of fixation and to produce as consistent ultracytochemical detection of cytochrome oxidase as possible, weak glutaraldehydeparaformaldehyde was employed as a fixative for 30 min fixation, according to Anderson et al (3).…”

“…The frozen sections were then thawed in 5% sucrose-0.1 M Hepes buffer in order to facilitate penetration of chemicals such as diaminobenzidine and cytochrome c into mitochondrial membranes, according to Kugler et al (13). All sections were then incubated for 120 min or 180 min at 37°C.…”

“…Also a methodological study of the cytochemical reactivity of cytochrome oxidase was included since fixation, incubation time and permeability of reagents during incubation affect the reactivity of the enzyme (3,7,13,23). et al…”