Showed Reaction Product Research Articles

Histochemical localization of zinc in the retina cells of gilthead sea bream (sparus aurata) fed different presentations of zinc.

To describe the distribution of zinc in the retina of a representative marine fish species and to determine whether the intracellular deposition amount correlates with the presentation of the zinc included in the practical diets (organic, inorganic, encapsulated, fish meal, and Control diet), we examined the precise localization of endogenous zinc in the gilthead sea bream (Sparus aurata) retina by autometallography. As observed by light microscopy, reaction products were widely distributed throughout the retina, including the outer segments of photoreceptors, except in the nuclear layers. Differing from other species previously studied, zinc depositions were not different between the outer and inner retina, and the retinal ganglion cell layer showed reaction products with a characteristic disposition surrounding the neuronal soma. An additional finding in this species was the rich disposition around photoreceptors, so abundant that it outlines the shape of the rods and cones. With regards to the diet, the zinc organic formulation was able to produce a higher amount of precipitates, followed by fish meal and encapsulated zinc diets. The inorganic and the Control diet produced a basal zinc deposition in the same layers above mentioned although less evident and similar to that determined in other species fed with non-supplemented diets.

Degradation of 2,4-dichlorophenol and coupling into humic matter by oxidative biomimetic catalysis with iron-porphyrin

We evaluated the activity of a water-soluble iron-porphyrin as catalyst of an oxidative reaction of 2,4-dichlorophenol (2,4-DCP) in the presence of humic molecules, whereby the phenol disappearance should proceed through self-olygomerization and co-polymerization within humic matter. The catalyzed coupling reaction was conducted with H2O2 as oxidizing agent with and without humic matter (HA) and followed by High Performance Liquid Chromatography (HPLC), upon incubation in either dark or daylight conditions. UV- and Fluorescence-detected signals of reaction products separated by HPLC were collected and structurally characterized by GC–MS. The 2,4-DCP degradation by the catalyzed oxidative reaction without addition of HA was complete in both incubation conditions and showed reaction products such as dehalogenated and hydroxylated phenols, and dimers/trimers of the dechlorinated phenol. With the presence of HA, 2,4-DCP was degraded in the dark by only 75% of initial content, whereas degradation was again complete in daylight, which showed similar reaction products as in the dark. These results reveal that the catalyzed oxidative reaction in the presence of HA was less efficient in the dark, possibly because 2,4-DCP was protected from oxidation by absorption into humic hydrophobic domains. Conversely, photo-oxidation combined to chemical oxidation to enhance the coupling reactivity in daylight conditions, most likely through co-polymerization of 2,4-DCP in humic molecules. This work confirms the potentiality of an oxidative coupling reaction catalyzed by iron-porphyrin in co-polymerizing halogenated pollutants into humic matter and detoxifying the environment.

Vascularization of the lateral line organ in the Atlantic cod: involvement of the secondary vascular system

AbstractSections of the lateral line organ, primary and secondary blood vessels and skin from the Atlantic cod Gadus morhua, Linnaeus 1758, were examined by light and transmission electron microscopy (TEM). The lateral line organ showed a structural analogy to the semicircular canals of the mammalian inner ear. A pericanalicular sinus (PCS), a canal of very loose connective tissue, surrounded the lateral line canal (LLC), separated by a multilayered epithelial wall. Located dorsal and ventral to the lateral line organ secondary vessels of capillary dimensions were found in association with the PCS. TEM of the wall of these dorso‐ventral vessels showed single tight junction contacts between the endothelial cells, allowing paracellular fluid exchange between the secondary vascular system and the PCS, an indication supported by horseradish peroxidase (HRP) tracer experiments, which showed reaction products in the PCS within 2 h after injecting HRP into the systemic circulation. The multilayered epithelial wall of the LLC showed multiple tight junctions between cells, making this boundary permeable only through transcellular transport.

Acidification of the parasitophorous vacuole containing Toxoplasma gondii in the presence of hydroxyurea

Toxoplasma gondii multiplies within parasitophorous vacuole that is not recognized by the primary no oxidative defense of host cells, mainly represented by the fusion with acidic organelles. Recent studies have already shown that hydroxyurea arrested the intracellular parasites leading to its destruction. In the present work we investigated the cellular mechanism involved in the destruction of intracellular Toxoplasma gondii. Fluorescent vital stains were used in order to observe possible acidification of parasitophorous vacuole-containing Toxoplasma gondii in presence of hydroxyurea. Vero cells infected with tachyzoites were treated with hydroxyurea for 12, 24 or 48 hours. Fluorescence, indicative of acidification, was observed in the parasitophorous vacuole when the cultures were incubated in presence of acridine orange. LysoTracker red was used in order to determine whether lysosomes were involved in the acidification process. An intense fluorescence was observed after 12 and 24 hours of incubation with hydroxyurea, achieving it is highly intensity after 48 hours of treatment. Ultrastructural cytochemistry for localization of the acid phosphatase lysosomal enzyme was realized. Treated infected cultures showed reaction product in vesicles fusing with vacuole or associated with intravacuolar parasites. These results suggest that fusion with lysosomes and acidification of parasitophorous vacuole leads to parasites destruction in the presence pf hydroxyurea.

LocaliZation of metabotropic glutamate receptor type 2 in the human brain

Metabotropic glutamate receptors are a heterogeneous family of G-protein-coupled receptors that are linked to multiple second messenger systems to regulate neuronal excitability and synaptic transmission. To elucidate the physiological role of these receptors in human central nervous system function and dysfunction at the receptor protein level requires the use of selective antibodies to determine the phenotype of cells expressing particular receptor subtypes. To this end the present study has examined the regional and cellular localization of the metabotropic glutamate type 2 receptor protein in selected human brain regions. After epitope prediction, antibodies have been generated against a short synthetic peptide corresponding to amino acid residues located in the putative intracellular carboxy-terminus and subsequently applied to an immunohistochemical investigation. Antibodies specifically detected the type 2 receptor in transfected mammalian cells and also recognized a major band of 98,000 mol. wt in western blots of human brain tissue membranes. At the light microscope level immunohistochemical studies have demonstrated that type 2-like immunoreactivity was widely distributed in the human brain, being characterized by the presence of a strong immunoreaction in multiple cortical regions, and in structures comprising the basal ganglia, to include the caudate nucleus, putamen, globus pallidus, substantia nigra and subthalamic nucleus. In the hippocampal formation, immunoreactivity was predominant in selective cell layers of both the dentate gyrus and cornu ammonis, the subicular complex and entorhinal cortex. In the thalamus, multiple subnuclei showed reaction product. In the cerebellar cortex, immunoreactivity was expressed in a number of cell layers and cell types. Furthermore, using double immunofluorescence we confirmed that the type 2 receptor is a product of normal resting astrocytes in the cerebral cortex in particular. This antibody provides a new immunological tool with the potential to evaluate the distribution of human metabotropic glutamate receptor 2 protein in other brain regions and in human central nervous system diseases.

Transient expression of NADPH-diaphorase/nitric oxide synthase in the paratenial nucleus of the rat thalamus

The distribution pattern of nitric oxide synthesizing neurons was studied in the paratenial nucleus throughout the rat development using the NADPH-diaphorase (ND) histochemical method and nitric oxide synthase (NOS) immunocytochemistry. The onset of ND/NOS activity in the paratenial nucleus was detected in the postnatal life day 1. Until the postnatal stage 4, a quick increase in the number and staining intensity of the ND/NOS positive neurons was observed. From postnatal day 4 to postnatal day 6, these variations continued slowly, whereas an increase in the neuronal size was evident. In these stages, densely packed ND/NOS-labeled neurons were observed. From stages 6 to 10, the ND/NOS-positive elements demonstrated similar number, size, and staining intensity. These cells had medium size, variable morphology and showed reaction product in the cell bodies and, at most, their proximal dendrites. After postnatal day 10, a quick decrease in the staining intensity and in the number of ND/NOS-labeled elements was detected, although no changes were observed in their morphological characteristics. Postnatal day 15 was the last developmental stage studied in which ND/NOS-positive elements were observed. Finally, the paratenial nucleus did not present ND/NOS-positive elements in adult animals. This transient expression of the ND/NOS-activity suggests a role of nitric oxide in the reorganization of the paratenial nucleus during the first postnatal fortnight.

Human Epithelial Related Antigen (hERA) in Salivary Glands and Their Tumors: Immunohistochemical Observations.

The expression of human epithelial related antigen (hERA), a 40 kD transmembrane glycoprotein assigned to one of the small cell lung cancer antigen was evaluated in normal salivary glands and their tumors using immunohistochemical methods.In normal salivary gland acini, hERA was seen in lateral borders and basal plasma membrane of serous cells of the parotid and submandibular glands and in the basal layer of mucous cells of the sublingual and minor oral glands, the luminally projecting cell membrane being consistently unreactive. In ductal segments, hERA was seen in basal infoldings and lateral borders of striated duct cells, excretory ducts or interlobular ducts and the ductal basal cells in particular, were devoid of hERA immunostaining.Salivary pleomorphic adenoma showed luminal borders of tubulo-ductal structures immunoreactive for hERA whereas the nonluminal or the neoplastic myoepithelial cells were consistently unreactive. A similar pattern of immunoreactivity was observed in Warthin's tumor where lateral borders of luminally located tall columnar tumor cells showed reaction products whereas the basal cells were unreactive. Mucoepidermoid carcinoma showed hERA in epidermoid and intermediate tumor cells, but not in mucous secreting cells. In adenoid cystic carcinoma reaction product for hERA was seen in a limited number of lumen-lining cells and again the non-luminally located neoplastic myoepithelial cells were unreactive. The results of the present study suggest that hERA may be a useful marker to detect luminal cell components in salivary tumor and, apart from the neoplastic myoepithelial cells or their counterparts, these luminally located cells may have a potential implication in salivary gland tumorigenesis.

Ultrastructural localization of the plasmalemmal calcium pump in cerebellar neurons.

In a previous study, fluorescence labeling of a plasmalemmal ATPase protein with the 5F10 monoclonal antibody revealed prominent antigen in the cerebellar molecular layer surrounding the somata and dendrites of Purkinje cells. In the present study, this antibody labeled with silver enhanced nano-sized gold particles on semithin plastic sections revealed a clearly demarcated plasma membrane outlining the somata and entire dendritic arbors of Purkinje cells including their spines. Ultrastructural analysis of horseradish peroxidase preparations showed reaction product along the plasmalemma and extending on to the sub-plasmalemmal endoplasmic reticulum. In the granular layer, somata of granule cells were reactive, as were their dendritic extensions into glomeruli where reactive claws surrounded voids formed by mossy fiber rosettes. Somata and dendrites of cerebellar nuclear cells also had reactive zones that were limited to the plasma membrane and a narrow zone of the sub-plasmalemmal endoplasmic reticulum. Comparative labeling of this protein and P channel protein revealed similar plasmalemmal locations. This study shows that a specific calcium ATPase pump protein is located on the plasmalemma of certain types of cerebellar neurons. The ultrastructural distribution of calcium pump and P channel antibodies occurred in punctate sites along the plasma membrane of dendrites and spines of Purkinje cells. The close association between P-type calcium channels and the plasma membrane calcium pump is consistent with rapid extrusion of intracellular calcium from neurons endowed with large numbers of voltage-gated calcium channels.

The cellular localization of malonyl-coenzyme A decarboxylase in rat brain.

Malonyl-coenzyme A (CoA) decarboxylase (E.C.4.1.1.9) activity in brain is low but steadily increases after birth. The main physiological role of this mitochondrial enzyme is thought to be the stabilization of malonyl-CoA levels which change very little with brain growth. In an effort to visualize malonyl-CoA decarboxylase by immunocytochemistry, and to determine its developmental changes, the enzyme was purified by an efficient small-scale procedure involving isolation of mitochondria, extraction at high ionic strength, isoelectric focusing, column chromatography, and preparative polyacrylamide gel electrophoresis. The enzyme from brain showed the same apparent molecular weight (160 kDa) and was immunoreactive with antisera raised against malonyl-CoA decarboxylase from liver. Immunocytochemistry revealed early and extensive labeling of hepatocytes in rat liver but only delayed visualization in the brain. Most nerve cells of the cerebral cortex and many microglia were stained but the neurons of the cerebellar cortex did not become reactive. Golgi epithelial cells and their processes, the Bergmann glia, also showed reaction product.

Transformation from SAA2-fibrils to AA-fibrils in amyloid fibrillogenesis: in vivo observations in murine spleen using anti-SAA and anti-AA antibodies.

Early amyloid fibrillogenesis from serum amyloid A protein (SAA) has been observed in the murine spleen after an injection of casein-Freund's complete adjuvant in the presence of amyloid enhancing factor, using anti-SAA C-terminal (anti-SAA) and anti-amyloid A (AA) antibodies. In Western immunoblotting of sera, both SAA1 and SAA2 reached a maximum after 24 h and began to decrease after 48 h. In spleen extracts, SAA2, but not SAA1 or AA, was found from 48 h, when amyloid was first deposited in the marginal zone. Electron microscopic immunohistochemistry of this stage showed reaction products from SAA in the marginal zone as fine granules along the cell membrane of mononuclear cells and focal intercellular aggregates, which contained fine fibrils originating from the cell membrane. Amyloid nodules, surrounded by mononuclear cells, developed from this stage. In the nodules, fibrils were positive for anti-SAA only in the vicinity of the cell membrane, while anti-AA stained fibrils throughout. Our hypothesis for fibrillogenesis is thus as follows: Serum SAA2 is specifically deposited on mononuclear cells in the marginal zone and polymerized extracellularly into fibrils, retaining its antigenicity (SAA2 amyloid fibrils); these fibrils are then processed to AA amyloid fibrils in situ by cleavage of the C-terminal portion of SAA2.

Ultrastructural localization of urease of Helicobacter pylori.

Helicobacter pylori urease was characterized by means of an enzyme histochemical electron microscopic technique. Ultrastructural analysis revealed no urease activity in one strain; in seven H. pylori strains (43.75%), urease activity was associated with the cell membrane. Eight strains (50.0%) showed reaction product located within the cytoplasm. Urease activity showed no correlation with localization of activity. Our results demonstrate that H. pylori urease is not uniform in all H. pylori strains, and differences in activity and localization of urease activity may account for different virulence activities.

Subcellular distribution of serotonin in the lamprey spinal cord

The subcellular distribution of serotonin (5-hydroxytryptamine; 5-HT) in the lamprey ( Ichtyomyzon unicuspis, Lampetra fluviatilis) spinal cord was investigated by using ultracentrifugation on continuous density gradients combined with an electron microscopic analysis of the gradients and of immunostained tissue. Endogenous 5-HT was analyzed by high-performance liquid chromatography with electrochemical detection. After differential centrifugation, the highest levels of 5-HT were found in the particulate fractions. After ultracentrifugation of lysed synaptosomal fractions on continouus sucrose gradients and the subsequent sedimentation of the individual fractions, 5-HT showed a biphasic distribution in the gradient. The two peaks corresponded to 0.30–0.40 M and 0.85–1.05 M sucrose. Electron microoscopy of intact tissue showed that some of the boutons were strongly immunoreactive to 5-HT with dense precipitates over large granular vesicles. The area around these large vesicles, however, also showed reaction product. Large granular vesicles could be clearly distinguished in the immunostained axonal varicosities. In tissue not processed for 5-HT immunoreactivity it was seen that the varicosities contained not only large dense-cored vesicles, but also small agranular vesicles. An electron microscopical analysis of the subcellular fractions revealed that the fraction corresponding to the “light” 5-HT peak contained numerous vesicular structures, which in most cases were electron lucent. In the “heavy” fractions, nerve ending particles containing vesicles of various sizes were observed. The results suggest that 5-HT in the lamprey spinal cord may be distributed in more than one subcellular compartment wwhich, apart from the cytosol, possibly corresponds to small and large synaptic vesicles.

Cochlear IgG in the C3H/ lpr autoimmune strain mouse

The inner ear of the C3H/ lpr autoimmune strain mouse was evaluated to identify potential mechanisms by which systemic autoimmune disease interferes with auditory function. The inner ears were immunohistochemically stained for IgG at ages before (2 months) and after (6–10 months) autoimmune disease onset and compared to age-matched nonautoimmune C3H/HeJ controls. Immunoreactivity for IgG was not seen in the 2 month C3H/ lpr autoimmune mice or in either age group of the C3H/HeJ controls. On the other hand, all older C3H/ lpr mice showed reaction product in the vessels of the cochlea, particularly the stria vascularis and bony capsule. Less frequent sites of staining were the geniculate ganglion, marrow cavities of the bony capsule, tensor tympani muscle, and on one occasion, a hair cell of the organ of Corti. These findings indicate that IgG is widespread within the cochlea and its vessels during systemic autoimmune disease and not directed against any specific sensorineural structure. This suggests a generalized or indirect mechanism whereby such systemic disease affects the inner ear.

Cytochrome oxidase activity as a measure of synaptogenesis by multifunctional neuron L7 of Aplysia.

Neuron L7 of the marine mollusc, Aplysia californica, is unique in that it innervates five different target tissues in the animal. We show that when L7 is grown in vitro with two of these targets, that is, muscle cells isolated from the auricle or the gill vein, newly formed L7 neurites contact the muscle cells. Chemical synapses are formed since intracellular stimulation of L7 elicits contraction of individual muscle cells. Interestingly, auricle muscles are also innervated by neuron RBhe and co-cultures of RBhe and auricle muscle cells also exhibit synapse formation. To explore the molecular basis for synaptogenesis between L7 and its targets, it would be useful to quantify the extent of synapse formation in vitro, that is, to determine how many muscle cells can be innervated by a single L7. We show that this can be attained by staining for cytochrome oxidase activity. Cultures of auricle and gill vein muscles were exposed to the appropriate neurotransmitter in order to elicit contraction. The cells were then fixed and stained. In both cases, only cells that contracted were stained and electron microscopy showed reaction product associated with the cristae of mitochondria. When this procedure was applied to cultures of L7 and muscle cells, 38 +/- 2.8% (S.E.M.; n = 7) of the cells on the neurites were stained and therefore responded to L7 stimulation. Thus, part of the L7-RBhe circuit can be assembled in vitro and the extent of synaptogenesis can be accurately quantitated.

The Determination of Interfacial Structure and Phase Transitions in Al/Cu and Al/Ni Interfaces by Means of Surface Extended X-Ray Absorption Fine Structure

ABSTRACTSurface extended x-ray absorption fine structure (SEXAFS) was used to investigate the interfacial conditions of Al/Cu and Al/Ni shallow buried interfaces. Previous studies using glancing angle extended x-ray absorption fine structure, x-ray reflectivity, photoemission, and SEXAFS produced conflicting results as to whether or not the interfaces between Al and Cu and Al and Ni were reacted upon room temperature deposition. In this study polycrystalline bilayers of Al/Cu and Al/Ni and trilayers of Al/Cu/Al and Al/Ni/Al were deposited on tantalum foil at room temperature in ultra high vacuum and analyzed to evaluate the reactivity of these systems on a nanometer scale. It became overwhelming apparent that the interfacial phase reactions were a function of the vacuum conditions. Samples deposited with the optimum vacuum conditions showed reaction products upon deposition at room temperature which were characterized by comparisons to standards and by least squares fitting to be CuAl2 and NiAl3 respectively. The results of this study showed that the reacted zone thicknesses were readily dependent on the deposition parameters. For both Al on Cu and Al on Ni as well as the metal on Al conditions 10A reaction zones were observed. These reaction zones were smaller than that observed for bilayers of Al on Cu (30Å) and Al on Ni (60Å) where deposition rates were much higher and samples were much thicker. The reaction species are evident by SEXAFS, where the previous photoemission studies only indicated that changes had occurred. Improved vacuum conditions as compared to the earlier experiments is primarily the reason reactions on deposition were seen in this study as compared to the earlier SEXAFS studies.

Localization of cathepsin B in normal and hyperplastic human prostate by immunoperoxidase and protein A‐gold techniques

Cathepsin B, a lysosomal cysteine protease, was localized in normal prostate and benign prostatic hyperplasia (BPH) using immunoperoxidase and protein A-gold techniques. Our objective was to determine whether cathepsin B was involved in the prostatic epithelium affected by nodular hyperplasia. All samples were collected immediately after prostatectomy. Immunohistochemical studies showed that the enzyme was expressed in the supranuclear cytoplasm of columnar cells and in numerous basal cells of normal and BPH acini. The strongest localization of cathepsin B occurred in acinar basal cells; hence, it is possible that cathepsin B could be useful as a marker for such cellular elements. Stromal macrophages showed reaction products, but lymphocytes and neutrophils did not. In both normal and hyperplastic glands, the enzyme was localized by gold particles in lysosomes, secretory granules, and vacuoles of columnar epithelial acinar cells. Immunoelectron microscopic study also showed the presence of cathepsin B in the heterochromatin (condensed chromatin) and nuclear membranes of columnar and basal cells, but not in euchromatin or nucleoli. At present, the function of cathepsin B in the nuclei of basal and columnar cells remains unknown. However, the cathepsin B in the cytoplasmic compartment might be associated with the lysosomal function of the cells. The role of cathepsin B as a marker for basal cell participation in the development of prostatic lesions should be studied further.

Characterization of ischemia-induced loss of epithelial polarity

Total renal ischemia for various time intervals (0-50 min) resulted in the rapid and duration-dependent redistribution of polarized membrane lipids and proteins in renal proximal tubule cells. Following only 15 min of ischemia, apical membrane enrichment of NaK-ATPase, normally a basolateral membrane (BLM) enzyme, had increased (1.6 +/- 0.6 vs. 2.9 +/- 1.2, P less than 0.01). In vivo histochemical localization of NaK-ATPase showed reaction product throughout the apical microvillar region. PTH-stimulatable adenylate cyclase, another BLM protein, was also found in ischemic but not control apical membrane fractions. One dimensional SDS-PAGE showed four bands, present in control BLM and ischemic apical membranes, which could not be found in control apical membrane fractions. Immunohistochemical localization of leucine aminopeptidase (LAP) showed the enzyme was limited to the apical domain in control cells. Following ischemic injury (50 min), LAP staining could be seen within the cell and along the BLM. Following 24 hr of reperfusion, the BLM distribution of LAP was further enhanced. With cellular recovery from ischemic injury (5 days), LAP was again only visualized in the apical membrane. Duration-dependent alterations in apical and BLM lipids were also observed. Apical sphingomyelin and phosphatidylserine and the cholesterol-to-phospholipid ratio decreased rapidly while apical phosphatidylcholine and phosphatidylinositol increased. Taken together, these results indicate renal ischemia causes rapid duration-dependent reversible loss of surface membrane polarity in proximal tubule cells.

Light and electron microscopic studies on the synovial membrane in Reiter's syndrome. Immunocytochemical identification of chlamydial antigen in patients with early disease.

Studies by light microscopy on synovium obtained from 11 patients with Reiter's syndrome during the first month of an episode showed proliferation of synovial lining cells, polymorphonuclear neutrophils among the synovial lining cells, increased surface fibrin, and vascular congestion. Biopsy specimens taken later showed vascular congestion and still proliferated synovial lining cells, fewer polymorphonuclear neutrophils in some, and a tendency toward increased infiltration with lymphocytes and plasma cells. Electron microscopy of samples from 8 patients during the first month of disease activity showed occlusion of vessels by platelets in 4, and fibrin or dense granular material in the vessel walls in 4. Five of the patients with arthritis of less than 4 weeks duration had unidentified intracellular and extracellular particles; some of these were highly suggestive of Chlamydia. No such particles were noted in samples from patients with more chronic cases. Using an antibody to Chlamydia trachomatis and the peroxidase-antiperoxidase technique, immunocytochemistry showed reaction product in synovial macrophages in 2 patients with arthritis of less than 4 weeks duration, but not in the 1 patient studied who had more chronic disease. These studies provide support for dramatic synovial vascular injury consistent with that caused by endotoxin and the presence of chlamydial antigen in synovial macrophages, at least in the early phases of synovitis.

Cytochemical localization of Ca 2+-ATPase activity in peripheral nerve

We used an electron microscopic cytochemical method to determine the localization of Ca 2+-ATPase in rat peripheral nerve. We found that reaction product occurred along most cytoplasmic membranes in the dorsal root ganglia (DRG). Unmyelinated axons demonstrated reaction product on the axolemma diffusely along their length. Myelinated fibers, in contrast, had reaction product limited to the axolema in the paranodal region. Internodal axolemma never showed reaction product and nodal axolemma was only occasionally stained, usually in sections reacted for the maximum times. Schwann cell plasma membranes uniformly showed reaction product. The restricted localization of Ca 2+-ATPase to the paranodal region of myelinated fibers suggests that calcium efflux may occur principally at those sites.

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.