DAB Reaction Product Research Articles

Abstract 354: Inducible Disruption of Endothelial Hyaluronan Synthase-2 Expression in Adult Mice Reveals a Direct Involvement of Glomerular Endothelial Surface Hyaluronan in Development of Albuminuria and Renal Injury

We demonstrated that infusion with hyaluronidase of C57Bl/6 mice for 4 wks to chronically reduce EC surface hyaluronan expression resulted in glomerular albumin passage and podocyte injury. However, with systemic hyaluronan degradation it precludes definitive conclusions on cellular-specific effects. Here we investigated the hypothesis that it is specifically the structure and integrity of the glomerular EC glycocalyx that determines whether or not albumin passage across the capillary wall occurs, and subsequently protects against development of kidney injury. To this end, we generated a conditional EC specific hyaluronan synthase2-knock out (HAS2–KO) mouse model. 8 Week old B6.VECad-creERT2.has2f/f.Rosa26-tdTomato were injected i.p. for 5 consecutive days with 2mg/0.2μL tamoxifen (control B6.VECad-creERT2.Rosa26-tdTomato) to induce an endothelial specific deletion of has2. Compared to ctrl, these mice show that 4 wks after induction, weight gain was significantly more increased (mean ± SEM; 2.9 ± 0.7 [n=8] vs. 5.2 ± 0.6g [n=11], p=0.02). With prominent endogenous albumin passage over the barrier, visualized as a DAB reaction product in electron microscopy after anti-albumin-HRP antibody incubation of 2%PFA fixated tissue slides, visible 2 wks after induction, a significant (p=0.036) increased ACR (36.0 ± 1.9 [n=8] vs. 57.7 ± 7.9 [n=11]) was found only at wk 4. During this period no significant change in BP (85 ± 2/114 ± 2 [n=8] vs. 90 ± 4/119 ± 4mmHg [n=10], diastolic/systolic) or in heart rate (712 ± 14 [n=8] vs. 747 ± 8beats/min [n=10]) was observed. Renal morphology revealed with comparable glomerular surface area (5342 ± 270 [n=3] vs. 5508 ± 250 μm2 [n=6]), significant increase of glomerular mesangial area (20.1 ± 0.9 [n=3]) vs. 38.4 ± 1.2% [n=6], p<0.01) and capillary surface area (16.6 ± 1.0 [n=3]) vs. 22.8 ± 0.7% [n=6], p=0.046). In conclusion, the data so far indicate that an intact glomerular EC-ESL is essential for glomerular integrity and prevention of albumin filtration is Key to preserving local quiescence of mesangial cells and podocytes. The conditional EC specific HAS2–KO mouse seems to be a valid model to assess the role of ESL in glomerular permeability barrier and vascular induced glomerular changes.

Regional specialization in pyramidal cell structure in the limbic cortex of the vervet monkey (Cercopithecus pygerythrus): an intracellular injection study of the anterior and posterior cingulate gyrus

The pyramidal cell phenotype varies quite dramatically in structure among different cortical areas in the primate brain. Comparative studies in visual cortex, in particular, but also in sensorimotor and prefrontal cortex, reveal systematic trends for pyramidal cell specialization in functionally related cortical areas. Moreover, there are systematic differences in the extent of these trends between different primate species. Recently we demonstrated differences in pyramidal cell structure in the cingulate cortex of the macaque monkey; however, in the absence of other comparative data it remains unknown as to whether the neuronal phenotype differs in cingulate cortex between species. Here we extend the basis for comparison by studying the structure of the basal dendritic trees of layer III pyramidal cells in the posterior and anterior cingulate gyrus of the vervet monkey (Brodmann's areas 23 and 24, respectively). Cells were injected with Lucifer Yellow in flat-mounted cortical slices, and processed for a light-stable DAB reaction product. Size, branching pattern, and spine density of basal dendritic arbors were determined, and somal areas measured. As in the macaque monkey, we found that pyramidal cells in anterior cingulate gyrus (area 24) were more branched and more spinous than those in posterior cingulate gyrus (area 23). In addition, the extent of the difference in pyramidal cell structure between these two cortical regions was less in the vervet monkey than in the macaque monkey.

DENDRITIC BRANCHING PATTERNS OF PYRAMIDAL CELLS IN THE VISUAL CORTEX OF THE NEW WORLD MARMOSET MONKEY, WITH COMPARATIVE NOTES ON THE OLD WORLD MACAQUE MONKEY

The basal dendritic arbors of 442 supragranular pyramidal cells in visual cortex of the marmoset monkey were compared by fractal analyses. As detailed in a previous study,1 individual cells were injected with Lucifer Yellow and processed for a DAB reaction product. The basal dendritic arbors were drawn, in the tangential plane, and the fractal dimension (D) determined by the dilation method. The fractal dimensions were compared between cells in ten cortical areas containing cells involved in visual processing, including the primary visual area (V1), the second visual area (V2), the dorsoanterior area (DA), the dorsomedial area (DM), the dorsolateral area (DL), the middle temporal area (MT), the posterior parietal area (PP), the fundus of the superior temporal area (FST) and the caudal and rostral subdivisions of inferotemporal cortex (ITc and ITr, respectively). Of 45 pairwise interareal comparisons of the fractal dimension of neurones, 20 were significantly different. Moreover, comparison of data according to previously published visual processing pathways revealed a trend for cells with greater fractal dimensions in "higher" cortical areas. Comparison of the present results with those in homologous cortical areas in the macaque monkey2 revealed some similarities between the two species. The similarity in the trends of D values of cells in both species may reflect developmental features which result in different functional attributes.

Osmium as an Immunostain Enhancement

Abstract Since we do our paraffin-section DAB immunostaining in an EM lab, we often use osmium to darken the reaction product. We simply put a drop of our standard 1% cacodylate-buffered osmium tetroxide on each section in a hood and rinse it off in running tap water after about 2 seconds. It makes light DAB reaction product pretty dark brown, and adds a light brownish tint to the rest of the section. The reaction increases the contrast quite a bit.

Pituitary adenylate cyclase activating polypeptide (PACAP) is present in human and cat gastric glands☆

In the present work we have studied the occurrence of pituitary adenylate cyclase activating polypeptide (PACAP) in human and cat stomach mucosa using immunohistochemistry. As seen under a light microscope, there were many large rounded and ovoid cells that were PACAP immunopositive, mainly in the neck of the gastric glands of both species. The immunopositive material was predominant in the perinuclear area. The PACAP immunolabeling was specific because the preincubation of the antiserum with PACAP abolished the immunostaining. In human samples under electron microscope, the PACAP immunoreactive cells have shown the characteristics of parietal cells. In faintly stained cells, the localization of DAB reaction product was associated with the surface of the intracellular canaliculi. Cell labeling could not be observed besides parietal cells.

The cerebral perivascular cells.

This monograph reviews the literature and presents experimental data on the intracerebral presentation of antigen(s) to the immune system as a consequence of neuronal cell death. "Which cells are the antigen presenting cells (APC) of the brain?" is the main question of this book. The immune surveillance of the CNS occurs through specialized resident cells, which present (auto)antigen(s) to the immune system and thus initiate an (auto)immune response. There are four established prerequisites necessary to identify resident APC of the brain. First, the APC must be capable to phagocytose dead neurons. Second, in order to be recognized by T lymphocytes, these neuronophages must express Major Histocompatibility Complex (MHC) cells II glycoproteins on their surface. Third, in order to present (auto)antigen, the MHC class II-positive neuronophages must also be able to contact T lymphocytes. Fourth, in order to exert a stimulatory effect on T lymphocytes, the APC should be able to produce the cytokine interleukin-1 beta (IL-128 Mb). Three main tools were used to identify and characterize the APC of the brain. First, a lesion model was employed that yields a slowly progressing neuronal cell loss without disruption of the blood-brain barrier. This model consisted of resection of 10 mm of the facial nerve, which caused a slowly occurring neuronal death so that one year after resection the amount of facial neurons was about 44% of the control value. Second, neuronophages were labeled in vivo in situ via phagocytosis of the permanent fluorescent marker Fluoro-Gold (FG) from decaying pre-loaded facial motoneurons. Third, the FG-labeled neuronophages were immunocytochemically characterized with the new method "immunoquenching of fluorescence". Sections of the brainstem containing FG-labeled, i.e. fluorescent, neuronophages were incubated with a variety of primary antibodies, followed by avidin-HRP and DAB-nickel as a dark brown reaction product for bright-field microscopy. In the fluorescent mode this DAB reaction product selectively quenches the fluorescence of all immunopositive cells, i.e. only those neuronophages that do not bind to the primary antibody remain fluorescent. Combining FG-labeling of neuronophages with immunoquenching, a population of small round fluorescent cells was discovered, localized in the immediate vicinity of the motoneurons long after the neuronofugal migration of microglia. As the fluorescence of these cells was not quenched after a triple immunostaining with anti-neuronal-specific enolase, anti-GFAP and OX-42 (quenching all fluorescence from neurons, astroglia, and microglia), they seem to represent a new, immunologically unidentified neuronophage. Following this triple immunostaining, a broad panel of antibodies was tested to stain, quench fluorescence, and thus immunotype these enigmatic phagocytes. Only the monoclonal antibody ED2, the classical marker for perivascular cells, specifically stained the small round neuronophages. Although the perivascular cells are in the vicinity of the basal lamina of the cerebral vasculature, they must not be confused with the pericytes, which are not able to perform phagocytosis. In contrast, the perivascular cells are macrophages-ED2 recognizes an established macrophage membrane antigen. In addition, after neuronal injury a subset of the perivascular cells starts to synthesize MHC class II glycoproteins and IL-1 beta. Hence this population of cells seems to possess the complete machinery required for antigen presentation: They are macrophages, upregulate MHC class II molecules and IL-1 beta, and due to their anatomical location, have access to circulating T lymphocytes. What was still lacking, however, was a direct proof of neuronophagia. Our experiments provided this proof. (ABSTRACT TRUNCATED)

Anterograde and retrograde traffic between the rough endoplasmic reticulum and the Golgi complex.

The transfer of newly synthesized membrane proteins moving from the rough endoplasmic reticulum (RER) to the Golgi complex has been studied by electron microscopy in HEp-2 cells transfected with cDNAs for chimeric proteins. These proteins consist of a reporter enzyme, horseradish peroxidase (HRP), anchored to the transmembrane domains of two integral membrane proteins, the transferrin receptor and sialyl-transferase. The chimeras are distributed throughout the nuclear envelope, RER, vesicular tubular clusters (VTCs) and a network of tubules in the cis-Golgi area. At 20 degrees C tubules containing chimera connect the RER to the VTCs and to the cis-Golgi network. On transfer to 37 degrees C in the presence of dithiothreitol (DTT), the chimeras are seen to move from the RER and through the Golgi stack. With this temperature shift the direct connections with the RER are lost and free vesicles form; some of these vesicles contain HRP reaction product which is much more concentrated than in the adjacent RER while others lack reaction product entirely. In cells expressing SSHRPKDEL, DAB reaction product remains distributed throughout the RER, the VTCs, and the cis-Golgi network for prolonged periods in the presence of DTT and almost all of the vesicles which form at 37 degrees C are DAB-positive. Together these observations demonstrate that all three chimeras are transported from the RER to the cis-Golgi in free, 40-60-nm vesicles at 37 degrees C. They also suggest that the retrograde traffic which carries SSHRPKDEL back to the RER is probably mediated by vesicles with a similar morphology but which, in cells expressing membrane-anchored chimeras, lack detectable reaction product.

Ultrastructural demonstration of endothelial glycocalyx disruption in the reperfused rat heart. Involvement of oxygen free radicals.

To determine the effect of post-ischaemic reperfusion on the ultrastructure of the endothelial glycocalyx and the role of oxygen free radicals, isolated working rat hearts were subjected to 20 min ischaemia followed by 3 or 30 min of reperfusion. Ruthenium red and lanthanum chloride were used to delineate the endothelial glycocalyx, and histochemical manganese/diaminobenzidine (Mn+2/DAB) or iron/diaminobenzidine (Fe+2/DAB) techniques were applied to visualize superoxide and hydrogen peroxide in myocardial capillaries. We found that ischaemia alone led to only a slightly flocculent appearance of the glycocalyx and its disruption was not observed until the onset of reperfusion. Prolongation of reperfusion to 30 min had no further effect on the ultrastructure of the glycocalyx. The ultrastructure of endothelial cells was normal. The disruption of the glycocalyx correlated in time and place with the appearance of Mn+2/DAB and Fe+2/DAB reaction products on the luminal surface of endothelial cells. Treatment with 5 mM N-(2-mercaptopropionyl)-glycine (MPG), an .OH radical scavenger, starting before ischaemia prevented the disruption of the glycocalyx, while 100 mM 3-morpholinosydnonimine (SIN-1), capable of generating both NO and -O2 simultaneously when applied at the time of reperfusion, increased the mean density of capillaries positively stained with Mn+2/DAB and Fe+2/DAB, and caused substantial disruption of the glycocalyx and damage to endothelial cells, which was not prevented by MPG. Our results suggest that the onset of reperfusion is critical for injury to the endothelial glycocalyx. Most probably the hydroxyl radical derived from the Fenton reaction is responsible for this injury. Peroxynitrite and/or nitric dioxide, if present upon reperfusion, may also account for damage of the endothelial glycocalyx.

Immunohistochemical localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the adrenal medulla of the rat

Localization of PACAP in rat adrenal glands was examined by light and electron microscopic immunohistochemistry using a specific antiserum to PACAP 38, R0831. In the light microscopic study, PACAP immunoreactivity was observed in some cell groups in the medulla, but not in the cortex. In comparison with adjacent sections stained with antisera to catecholamine synthesizing enzymes, PACAP-positive cells were immunoreactive to tyrosine hydroxylase and dopamine beta-hydroxylase, but not to phenylethanolamine-N-methyltransferase, suggesting that they were coincident with noradrenaline secreting cells. In the electron microscopic study using the ABC method, DAB reaction products were diffusely distributed in the cytoplasmic matrix of PACAP-positive cells, without intense accumulation on the secretory granules. The splanchnic nerve terminals were PACAP negative. In postembedding immunohistochemistry, gold particles were localized diffusely in the cytoplasma, but not aggregated on the secretory granules. It was suggested that PACAP would localize in the cytoplasmic matrix of noradrenaline cells and stimulate the catecholamine synthesis and release in the adrenal medulla.

Differences between standard and high-sensitivity immunohistology in tissue sections — comparison of immunoperoxidase staining methods using computerized video image analysis techniques

High-sensitivity immunoperoxidase labelling can be achieved using heavy metal enhancement of the di-amino (DAB) reaction product. For example nickel chloride combined with DAB improves the sensitivity of the method approximately 7-10 fold. This allows detection of approximately 100-200 molecules on cell surfaces. This has an obvious advantage over standard non-enhanced DAB methods which detect 1000-2000 molecules under similar conditions. This study compares standard and nickel enhanced DAB immunoperoxidase staining of cells in tissue sections using video-image analysis (VIA) measurement techniques. VIA is an objective method of evaluation of immunoperoxidase staining of separated cells and for immunostained cells in tissue sections. Nickel enhancement of the DAB reaction product reveals more positively stained cells, with higher contrast over background, giving superior qualities for VIA analysis providing continuous, reproducible, and objective data for statistical analysis.

Localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the hypothalamus-pituitary system in rats: light and electron microscopic immunocytochemical studies.

The localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the hypothalamus-pituitary system in rats was examined in light and electron microscopic immunocytochemistry using a specific antiserum to synthetic PACAP 1-38 (R0831). In light microscopic study, intensely PACAP-immunostained perikarya were observed in the supraoptic and paraventricular magnocellular nucleus in the hypothalamus. In the median eminence, many immunoreactive nerve fibers were observed in the internal layer, but a few immunoreactive terminals were noticed in the external layer. In the pituitary gland, numerous immunoreactive nerve fibers were observed in the posterior lobe. In the intermediate lobe, moderately immunostained cells were observed, but in the anterior lobe no immunostained cells were noticed. In electron microscopic study, PACAP-immunoreactivity was examined by avidin-biotin peroxidase complex method. In the perikarya of the supraoptic and paraventricular magnocellular nucleus, DAB-reaction products were distributed diffusely in the cytoplasmic matrix, frequently attaching to the rough-surfaced endoplasmic reticulum. In the nerve terminals of the posterior lobe, reaction products were observed among the secretory granules, but sometimes upon them. In the cells of the intermediate lobe, reaction products were also distributed in the cytoplasmic matrix.

Localization of laminin in nephritic glomeruli as revealed by a quick-freezing and deep-etching method with immunohistochemistry.

The three-dimensional localization of laminin in rat glomeruli at the chronic phase of Masugi nephritis was investigated by a quick-freezing and deep-etching method combined with immunohistochemistry. Light-microscopically, laminin was localized in increased mesangial matrix and thickened glomerular basement membrane. The quick-freezing and deep-etching method revealed that the increased mesangial matrix, which was newly formed in axial portions and areas of mesangial interposition, was composed of fine fibrillar networks. They were revealed with the 3,3'-diaminobenzidine tetrahydrochloride (DAB) reaction products of peroxidase-labelled secondary antibody following anti-laminin antibody. However, these reaction products were not uniformly distributed in the newly formed matrix. Although the fibrils organizing lamina densa were also immunostained with anti-laminin antibody, the fibrils connected to mesangial cells, podocytes and endothelial cells had smaller amounts of DAB reaction products for laminin. These results indicate that one of the components of fibrils in the mesangial matrix and lamina densa is laminin, which is heterogeneously distributed in the newly formed matrix.

Estrogen receptive neurons in the preoptic area of the rat are postsynaptic targets of a sexually dimorphic enkephalinergic fiber plexus

The periventricular preoptic area (pePOA) is a sexually dimorphic component of the rat forebrain that contains a sexually dimorphic Met-enkephalin immunoreactive (ENK-ir) fiber plexus. This plexus is especially dense in the female while only scattered ENK-ir fibers are present in the pePOA of the male. Abundant estrogen receptive neurons are located in the pePOA of both the female and male. This experiment was conducted to determine if estrogen receptive neurons in the pePOA are postsynaptic targets of ENK-ir terminals. Double label ultrastructural localization of estrogen receptor (ER)-ir neurons and ENK-ir fibers was performed using the chromogens 3,3′,5,5′-tetramethylbenzidine (TMB) and diaminobenzidine tetrahydrochloride (DAB), respectively. TMB-stained ER-ir neurons contained electron dense crystalline spicules located predominantly in their nuclei. Flocculent DAB reaction product was distributed over membraneous structures in ENK-ir fibers and terminals. Numerous ER-ir neurons were present in the pePOA of the male and female. In females, many ENK-ir terminals, both synaptic and non-synaptic, contacted the perikarya of ER-ir neurons. In contrast, many fewer ENK-ir terminals made contact on ER-ir neurons in the male. Thus, these results provide morphological evidence that ENK-ir neurons can regulate ER-ir neurons in the pePOA. Moreover, because expression of the ENK-ir pePOA fiber plexus is estrogen-sensitive in the female, these results suggest strongly that estrogen may regulate these neurons both pre- and postsynaptically. Finally, these results provide additional evidence for the involvement of the sexually dimorphic pePOA ENK-ir fiber plexus in the control of estrogen-mediated function in the female.

Use of anti-horseradish peroxidase antibody-gold complex in the ABC technique.

We report a modification of the avidin-biotin-peroxidase complex (ABC) technique for the light and electron microscopic detection of antigens in tissue sections. An immunological approach was used instead of the DAB reaction to reveal ABC bound to antigen-antibody complexes. Affinity-purified polyclonal antibodies against horseradish peroxidase were complexed to particles of colloidal gold and applied for reaction with the horseradish peroxidase molecules of the ABC. For light microscopic immunolabeling, the signal produced by the anti-horseradish peroxidase antibody-gold complex required silver intensification. The ABC immunogold reaction as compared with the standard ABC technique, in particular with silver intensification of the DAB reaction product, provided superior resolution in paraffin sections. Furthermore, section pre-treatment to block endogenous peroxidase activity could be omitted and no potentially hazardous substrate was used. The ABC immunogold reaction was successfully applied for electron microscopic immunolabeling on Lowicryl K4M thin sections. We propose that the ABC immunogold reaction is a useful alternative to the standard ABC technique and can be equally well applied to light and electron microscopy.

Parameters affecting imaging of the horseradish‐peroxidase‐diaminobenzidine reaction product in the confocal scanning laser microscope

Neurons intracellularly filled with biocytin and labelled with nickel-intensified diaminobenzidine (DAB/Ni) can be imaged on the confocal scanning laser microscope in order to obtain three-dimensional and optical section images of neurons. Intensification of the DAB reaction product with nickel was found to be crucial for obtaining a workable signal level. On the other hand, the high absorption of light by the reaction product severely attenuated the detection of structures lying directly underneath, and the intensity of the unattenuated laser used for imaging faded or damaged DAB/Ni reaction product. We have determined that reduction of the laser intensity combined with the use of proper objective lenses and non-laser-based imaging for preliminary adjustments of the specimen all work to reduce or eliminate damage, and also improve the image. These items must be kept in mind when imaging and analysing DAB-labelled structures in the laser-based confocal microscope.

Comparative study of lymphatics and bllod vessels.

Two methods were used to distinguish between lymphatic capillaries and blood capillaries at the light microscopic level: in one toluidine blue sections were embedded in epoxy resin following arterial perfusion-fixation, and in the other an immunohistochemical technique used basement membrane antisera (laminin, type IV collagen and fibronectin antisera) in indirect immunoperoxidase and immunogold-silver staining (IGSS) methods. The precise immunolocation of these basement membrane components around these capillaries was examined electron microscopically by pre-embedding immunoperoxidase and post-embedding immunogold techniques. The toluidine blue sections showed two types of capillaries: one with blue-stained lumens and the other with vacant lumens. Conventional electron microscopy revealed that capillaries with blue-stained lumens are lymphatic capillaries and capillaries with vacant lumens are blood capillaries by their fine structural characteristics. Immunohistochemical techniques using laminin or type IV collagen antiserum in both the immunoperoxidase and IGSS methods showed blood capillaries with continuous and linear reactions and lymphatic capillaries with absent or weak reactions beneath the endothelium. Immunostaining of fibronectin antiserum was observed nonspecifically around both types of capillaries and in connective tissue. Fine granular products in the IGSS method were in sharper contrast against the background and were more intensely stained and thinner than the DAB reaction products demonstrated by the immunoperoxidase method. Immunoelectron microscopy revealed greater immunoreactivity of laminin and type IV collagen antisera in the lamina densa around blood capillaries than in that around lymphatic capillaries. A striking feature was the presence of fibronectin antiserum in the intracellular organelles of blood capillary endothelium and on the surface of fibroblasts. In conclusion, toluidine blue staining following arterial perfusion-fixation is a useful technique for experimental pathologists, and the IGSS method using laminin and type IV collagen antisera is a practical light microscopic method of distinguishing between lymphatic capillaries and blood capillaries.

Papillomavirus antigen in the epidermoid cyst of the sole

An epidermoid cyst of the sole was studied immunohistochemically and ultrastructurally. Papillomavirus particles were present in the horny layer and in the upper layers of the epidermis of the cyst and within the acrosyringeal epithelium overlying the cyst. Thickened basal lamina-like structures similar to those found in the eccrine sweat duct tumors such as in cylindroma and eccrine spiroadenoma existed at the epidermal-dermal junction of the cyst wall. Carcinoembryonic antigen was immunohistochemically demonstrated in the center of the epidermoid cyst and the DAB reaction products took the shape of a circle resembling that of the sweat ducts in the horny layer.

Ganglion cells in the turtle retina contain the neuropeptide LANT-6

This study investigated the presence of the neurotensin-related hexapeptide, LANT-6, in retinal ganglion cells and their central projections in the turtle Pseudemys scripta elegans. Immunocytochemical techniques demonstrated that many of the cells in the ganglion cell layer of the turtle retina could be labeled with an antiserum specific for LANT-6. Radioimmunoassay and chromatographic analysis confirmed the presence of LANT-6-related peptides in retina, as well as brain. Several molecular forms of LANT-6 were observed, some larger than LANT-6. Characterization of the cells labeled in the ganglion cell layer in terms of their cell body size and their dendritic arborization patterns revealed that at least 6 specific LANT-6-positive cell types were present in the ganglion cell layer. Morphologically, the LANT-6-positive cells strongly resembled turtle ganglion cells, as previously described. In addition, two other lines of evidence supported this interpretation. First, double-label studies were performed in which retinal ganglion cells projecting to the tectum were retrogradely labeled by HRP injected into the tectum (using a cobalt chloride color-modified DAB reaction product) and immunocytochemically labeled with DAB using the antiserum against LANT-6. These double-label studies revealed that many of the LANT-6-positive cells in the ganglion cell layer in the portion of the retina labeled retrogradely by the HRP injection did project to the tectum. Within the retrogradely labeled portion of the retina, LANT-6-positive cells that were not labeled retrogradely, as well as neurons labeled retrogradely that did not contain LANT-6 were also observed. Second, the central projections of LANT-6-positive cells of the ganglion cell layer were examined by studying the effects of monocular enucleation on the distribution of LANT-6-positive fibers in the central projection targets of the turtle retina. Two to 8 weeks after enucleation, a substantial reduction in LANT-6-positive fibers was observed in all retinal target areas contralateral to the enucleated eye. Radioimmunoassay and chromatographic studies confirmed the presence of LANT-6-related peptides in the turtle brain and corroborated the reduction of LANT-6 observed in the contralateral tectum following monocular enucleation. Previous studies have demonstrated that LANT-6-related material is present in cells of the ganglion cell layer in a variety of vertebrates. The present results indicate that LANT-6 is in ganglion cells and that it may play a role in neurotransmission between retinal ganglion cells and their central target areas.

Characterization of cytoplasmic male sterility in Petunia hybrida and Zea mays. Localization and activity of cytochrome c oxidase

Anthers of male fertile, cytoplasmic male sterile (cms), and restored male fertile Petunia hybrida, are analyzed for cytochrome c oxidase (cox) activity in subsequent stages of microsporogenesis, and compared with anthers of male fertile, cms-S and cms-C Zea mays. The cox activity is determined in anther extracts and cytochemically. In petunia anthers, the first differences in cox activity occur from meiosis onward. However, at these stages, the initial symptoms of degeneration are already apparent. It is suggested that the decline in enzyme activity of the cms petunia anthers is the result rather than the cause of the non-formation of functional pollen. In maize anthers, the cox activity of sterile-type anthers is reduced in comparison with fertile-type anthers from premeiosis onward. There are also consistent cytochemical differences in the mitochondrial organization of cox activity between pollen of cms-S and male ferile maize anthers. In fertile-type mitochondria, the DAB reaction product indicating cox activity is localized in the cristae and within the space between the outer and inner limiting membranes of the organelles. In mitochondria of pollen of cms-S maize, cox activity is only observed between the outer and inner membranes of the mitochondria. The biochemical and cytochemical differences are observed at stages of development at which no structural signs of degeneration are apparent. The results suggest that cms in maize correlates with deviations in cytochrome c oxidase activity.

Benzidine dihydrochloride as a chromogen for single- and double-label light and electron microscopic immunocytochemical studies.

Although very sensitive chromogens have been adapted for localization of horseradish peroxidase in anterograde and retrograde tracing studies, they have not been successfully applied in immunocytochemical studies. This report describes a protocol which uses benzidine dihydrochloride (BDHC) as the chromogen for light (LM) and electron microscopic (EM) immunocytochemical studies. The protocol is comparable to that used for tetramethylbenzidine, except that the pH of the reaction is above 6.0. At the LM level, the BDHC reaction product is bluish-green and crystalline. Both the color and form of the product are readily distinguished from the reddish-brown DAB reaction product. LM double-labeling studies are therefore feasible. The use of BDHC also increases significantly the sensitivity of the immunoreaction. Higher fixative concentrations can be used, less detergent is necessary, and higher primary antibody dilutions are possible. By osmicating at 45 degrees C in an s-collidine buffer it is possible to preserve the soluble BDHC reaction product for EM analysis. Immunoreactive cells are particularly well labeled with this new protocol. The BDHC crystals are easily detected at the EM level and can be distinguished from flocculent DAB reaction product. This feature makes EM double-labeling studies possible.