Outer Segments Of Photoreceptor Cells Research Articles

The 220-kDa Rim Protein of Retinal Rod Outer Segments Is a Member of the ABC Transporter Superfamily

Outer segments of mammalian rod photoreceptor cells contain an abundantly expressed membrane protein that migrates with an apparent molecular mass of 220 kDa by SDS-gel electrophoresis. We have purified the bovine protein by immunoaffinity chromatography, determined its primary structure by cDNA cloning and direct peptide sequence analysis, and mapped its distribution in photoreceptors by immunocytochemical and biochemical methods. The full-length cDNA encodes a 2280-amino acid protein (calculated molecular mass of 257 kDa) consisting of two structurally related, tandem arranged halves. Each half consists of a hydrophobic domain containing six putative transmembrane segments followed by an ATP-binding cassette. A data base homology search showed that the rod outer segment 220-kDa protein is 40-50% identical in amino acid sequence to the ABC1 and ABC2 proteins cloned from a mouse macrophage cell line. Photoaffinity labeling with 8-azido-ATP and nucleotide inhibition studies confirmed that both ATP and GTP bind to this protein with similar affinities. Concanavalin A labeling and endoglycosidase H digestion indicated that the rod outer segment protein contains at least one carbohydrate chain. Immunocytochemical and biochemical studies have revealed that the 220-kDa glycoprotein is distributed along the rim region and incisures of rod outer segment disc membranes. From these studies we conclude that the 220-kDa glycoprotein of bovine rod outer segment disc membranes or Rim ABC protein is a new member of the superfamily of ABC transporters and is the mammalian homolog of the frog photoreceptor rim protein.

Effect of thyroidectomy on photoreceptor cells in adult rat retina

To evaluate the effect of thyroid hormone on the retina in the adult male rat, morphometric and autoradiographic studies were made. Four days after an intravenous pulse injection of L-[ 3H]-leucine into adult male rats, the eyeballs were removed and the autoradiogram made. Two weeks after thyroidectomy, the length of photoreceptor cell inner segments was significantly decreased, but that of the outer segments did not change. Daily supplement of T4 (100 μkg sc twice for 2 days) before L-[ 3H]-leucine injection did not restore the decrease in the length. The silver grains of radioactive material distributed in the outer segment were divided into 3 equal portions. Thyroidectomy significantly increased the number of grains in the basal portion near the inner layer indicating a reduction in the renewal rate of the outer segment, but the T4 supplement did not restore the reduction in the renewal rate. These findings suggest that thyroid hormone is required to maintain the rate of renewal of the photoreceptor cell outer segment but the hormone supplement did not rapidly restore the renewal rate.

Polyamines and ripening of photoreceptor outer segments in chicken embryos

Polyamines and their related monoacetyl derivatives were studied in rod outer segment (ROS) and cone outer segment (COS) of photoreceptor cells from chick embryo retina during eye development (7th–18th days). Putrescine was found to be necessary, in the second phase of retinogenesis, to sustain both ROS and COS differentiation and, after acetylation, γ-aminobutyric acid synthesis. On the other hand, spermidine and even more spermine intervene in the third phase of development when photoreceptors mature. Moreover, the presence of N1-acetylspermidine already at the 7th day indicates that in the outer segment of photoreceptor cells too, as in the whole retina, putrescine synthesis comes about by two pathways. One pathway involves ornithine decarboxylase; the other, spermidine/spermine N1-acetyltransferase and FAD-dependent polyamine oxidase activities that convert spermidine to putrescine via N1-acetylspermidine. These different biosynthetic pathways are probably also decisive in permitting γ-aminobutyric acid synthesis, which is very important in the ripening process of neural retina.

Expression and localization of inhibin α-subunit in rat retinal photoreceptor cells

To determine whether rat retinal photoreceptor cells produce inhibin, a molecule closely related to activin, a multifunctional growth factor in the transforming growth factor β superfamily (TGFβ), we have conducted immunohistochemistry using specific antibodies for inhibin which were raised against a synthetic N-terminal fragment of the α-subunit of inhibin. The mature inhibin molecule was identified at both the inner and outer segments of photoreceptor cells. To determine if messenger RNA for the α-subunit of inhibin is expressed in the retinal cells, both in situ hybridization with a specific probe and the reverse transcription-polymerase chain reaction (RT-PCR) technique with primers specific for the α-subunit of inhibin were used. Messenger RNA expression of the α-subunit of inhibin was detected by RT-PCR and localized in the photoreceptor cells as determined by in situ hybridization. In addition, the identity of the cDNA product of RT-PCR was verified with Southern analysis and DNA sequencing. The localization of mature inhibin protein and its corresponding message to photoreceptor cells suggest that inhibin may have a paracrine function in the retina, perhaps in the photoreceptor cells themselves.

Circadian rhythms of corneal mitotic rate, retinal melatonin and immunoreactive visual pigments, and the effects of melatonin on the rhythms in the Japanese quail.

We investigated circadian ocular rhythms in the Japanese quail, Coturnix coturnix japonica. The birds were placed under light-dark cycles (LD 12:12), constant light (LL) and constant darkness (DD), and the retinas were dissected out at four-hour intervals throughout 24 h. Following measurements were performed. (1) Melatonin content in the retina was measured by radioimmunoassay. It was low in light and several folds higher in darkness under LD 12:12. The rhythm continued in DD, but disappeared in LL. (2) Mitotic figures in the corneal epithelium were counted. Similar rhythms to the melatonin content were observed in the corneal mitotic rate with a slight phase delay. (3) The retinas were fixed at 4-h intervals and immunostained with anti-bovine rhodopsin serum and anti-chicken iodopsin monoclonal antibodies. The outer segments of photoreceptor cells were stained intensively throughout 24 h in LD 12:12, LL and DD. In contrast, the stainability of the locus close to the outer limiting membrane where the Golgi apparatus exists changed diurnally. Scores showing the ratio of cells with positive staining indicated high values from 4 h after the onset of light to the beginning of dark phase under LD 12:12. The values were high throughout 24 h in LL and intermediate or low in DD. (4) To investigate the effect of melatonin on the corneal mitotic rate and visual pigments at the Golgi region, melatonin was injected into one eye and saline into the contralateral eye. Melatonin induced a phase advance in the corneal mitotic rate under LD 12:12, but did not induce a rhythm under LL.(ABSTRACT TRUNCATED AT 250 WORDS)

Retinal Pigment Epithelium Cell Culture as a Model for Evaluation of the Toxicity of Tamoxifen and Chloroquine

The retinal pigment epithelium (RPE) removes the outer segments of photoreceptor cells by phagocytosis. We studied the effects of tamoxifen and chloroquine on the activity of the lysosomal enzymes N-acetyl-beta-glucosaminidase and cathepsin D in RPE in vitro to evaluate the possible eye toxicity caused by these drugs. The results show decreases in the activities of lysosomal enzymes after drug exposure. The enzymes tested seemed to be more sensitive to tamoxifen than to chloroquine. A profound decrease in the activities of the lysosomal enzymes only started at concentrations above therapeutic dose levels.

Taurine and GABA in the rat retina during postnatal development.

The content of taurine and the immunocytochemical localization of taurine and gamma-aminobutyric acid (GABA) in the rat retina during postnatal development are described. The rat retina is immature at birth; about two-thirds of the cells are undifferentiated neuroblasts, and the taurine content per retina is approximately one-seventh of the adult value. Shortly after weaning the adult morphology and taurine content are attained. Expression of taurine immunoreactivity (taurine-IR) accompanies differentiation; in some cell types (ganglion and horizontal cells) this expression is transient, while in others (photoreceptors, bipolar, and a subpopulation of amacrine cells) it persists into the adult state. At birth, taurine-IR is localized mainly in cells in the position of ganglion cells, especially in their axons within the nerve fiber layer. This reactivity is soon lost from the somata, and disappears from the axons by 10 days of age. At 2 days of age, taurine-IR appeared additionally in somata of amacrine cells flanking the forerunner of the inner plexiform layer, and in growth cone-like processes of photoreceptors. At day 6, taurine-IR was marked in photoreceptor cell inner and outer segments, and in horizontal cells and their lateral processes. Taurine-IR was lost from horizontal cells and most amacrine cells around day 10, and appeared in bipolar cells, where it remained, with that in photoreceptors, into adulthood. Particularly striking was taurine-IR in large synaptic terminal-like processes close to the ganglion cell layer which were first seen around day 16.(ABSTRACT TRUNCATED AT 250 WORDS)

Visual pigments in the pineal complex of the Japanese quail, Japanese grass lizard and bullfrog: Immunocytochemistry and HPLC analysis

We investigated localization of visual pigments in the pineal complex of Japanese quail, Japanese grass lizards and bullfrogs immunocytochemically by use of the antiserum against bovine rhodopsin (Rh-As) and monoclonal antibodies against chicken iodopsin (Io-mAb). We also analyzed retinoids, chromophores of visual pigments, by a high performance liquid chromatography (HPLC). The outer segments and cell membranes of some photoreceptor cells in the pineal organ of the Japanese quail exhibited immunoreactivity to Rh-As, but there are also many immunonegative cells. The number of immunoreactive cells among individuals varied. Immunoreactivity to Io-mAb was weak or did not exist. The HPLC analysis revealed peaks of 11-cis and all-trans isomers of retinal in the oxime extracts of the pineal organ of Japanese quail and chickens. In the pineal of Japanese grass lizards, the outer segments of some cells were immunopositive to Io-mAb, but there were no cells immunoreactive to Rh-As. The parietal eye exhibited a well-developed lens and photoreceptor cells, but the outer segments of photoreceptor cells were immunonegative to both Rh-As and Io-mAb. In bullfrogs, three types of cells were identified in both the pineal and frontal organ; (1) immunopositive to Rh-As, (2) immunopositive to Io-mAb and (3) immunonegative to either of the antibodies. In the pineal organ of bullfrogs, 11-cis and all-trans retinal and 11-cis 3-dehydroretinal were detected, and 11-cis and all-trans retinal were also detected in the frontal organ. We detected 11-cis and all-trans retinal in the ventral part of diencephalon including the hypothalamus. Thus, the chromophore is the same between the retinal and pineal visual pigments, but the expression of opsins is different between the retina and pineal complex, which probably reflects the different function of each organ.

Brain-type creatine kinase in photoreceptor cell outer segments: role of a phosphocreatine circuit in outer segment energy metabolism and phototransduction

Different isoforms of creatine kinase, an important enzyme of vertebrate energy metabolism, were localized in bovine photoreceptor cells, with particular emphasis on the identification and quantification of the brain-type isoform within the outer segment compartment. Using immunofluorescence and immunoelectron microscopy, brain-type creatine kinase was shown to be present in bovine photoreceptor cell outer and inner segments. The presence of this isoenzyme in rod outer segments was additionally confirmed by immunoblotting and immunolabeling of isolated rod outer segments. The content of creatine kinase in rod outer segments was quantified by measuring creatine kinase activity after membrane disruption with detergent. The ATP regeneration potential provided by the creatine kinase in isolated, washed bovine rod outer segments was 1.2 +/- (0.4) i.u. mg-1 rhodopsin. This value was calculated to be at least an order of magnitude larger than that necessary to replenish the energy required for cGMP resynthesis in rod outer segments, and high enough to regenerate the entire ATP pool of rod outer segments within the time span of a photic cycle. A mitochondrial creatine kinase isoenzyme was located within the ellipsoid portions of bovine rod and cone inner segments by immunofluorescence microscopy and, using immunogold staining, was specifically localized in the mitochondria clustered within bovine rod and cone inner segments. These results suggest that vertebrate photoreceptor cells contain a functional phosphocreatine circuit. Outer segment creatine kinase may play an important role in phototransduction by providing energy for the visual cycle, maintaining high local ATP/ADP ratios and consuming protons produced by enzymes located in the outer segment.

Light stimulates in vivo inositol lipid turnover in frog retinal pigment epithelial cells at the onset of shedding and phagocytosis of photoreceptor membranes

We have developed an experimental model to study in vivo inositol lipid metabolism in frog retinal pigment epithelial (RPE) cells, including the effect of light on phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. RPE cells were rapidly isolated after either brief light or dark periods. Light and electron microscopy showed complete detachment of the retina from the RPE cells, and that the RPE cell suspensions were devoid of photoreceptor cell outer segments. Frog tissues were labeled in vivo for 20 hr by intravitreal injection of [ 3H]inositol (4 μCi, 4μl per eye) within a 24-hr constant illumination period. Following 1 hr of darkness (priming period), frogs were intravitreally injected with LiCl (0·5 m, 4μl per eye) 15 min before the onset of either 30-min light stimulation or an additional 30 min of darkness (controls). In order to preserve endogenous inositol phosphate pools present after dark and light exposure, the RPE cells were harvested in the shortest time possible, at low temperatures (18–20°C), and in the presence of 10 m m LiCl. Total [ 3H]inositol-labeled water-soluble products (inositol plus inositol phosphates) were increased by 86% after 30 min of light. Inositol trisphosphate (IP 3) showed the highest accumulation (a 5·5-fold increase), followed by inositol bisphosphate (1·9-fold increase) and inositol monophosphate (1·4-fold increase). Free [ 3H]inositol also accumulated (2·8-fold increase), reflecting only a partial inhibition of phosphomonoesterase by LiCl. These changes were paralleled by a 12% decrease in 3H-labeled phosphatidylinositol with no significant difference in the labeling of polyphosphoinositides. This is the first time that IP 3 accumulation, correlated with a specific function in a cell type, has been demonstrated in vivo.

Highly sialylated N-CAM is expressed in adult mouse optic nerve and retina

The localization of the neural cell adhesion molecule (N-CAM) and its highly sialylated form, which is prevalent in young tissues and has therefore been called embryonic neural cell adhesion molecule, was studied in the developing and adult mouse optic nerve and retina immunohistologically and immunochemically. At embryonic and early postnatal ages, neuroblasts and young postmitotic neurons, Müller cells and astrocytes in the retina, and retinal ganglion cell axons and all glial cells in the optic nerve express highly sialylated neural cell adhesion molecule. Beginning with the third postnatal week, highly sialylated neural cell adhesion molecule disappears from retinal ganglion cell axons in the optic nerve and from neuronal cell bodies and processes in the retina. In addition, it is not detectable on oligodendrocytes in 3-week-old animals. However, highly sialylated neural cell adhesion molecule continues to be expressed in the adult optic nerve and retina by astrocytes and Müller cells. On these cells it is only absent from cell membranes contacting basal lamina. Weakly sialylated neural cell adhesion molecule, in contrast, is expressed by all cell types of retinal and optic nerve during development and in the adult. The loss of highly sialylated neural cell adhesion molecule from neurons and oligodendrocytes must therefore be considered as a cell type-specific conversion of the so-called embryonic to the adult form of neural cell adhesion molecule and does not simply reflect the disappearance of neural cell adhesion molecule from these cells. Weakly sialylated neural cell adhesion molecule, however, is absent from outer segments of photoreceptor cells and, as is the case for the highly sialylated form, from glial cell surfaces contacting basal lamina. Thus, the expression of highly sialylated neural cell adhesion molecule by pre- and postmitotic neurons and by oligodendrocytes is restricted mainly to the period of histogenetic events in retina and optic nerve, i.e. cell division, cell migration, dendritic and axonal growth and synaptogenesis. In addition to the observation that this form of neural cell adhesion molecule is less adhesive than the weakly sialylated, adult form, it is likely that highly sialylated neural cell adhesion molecule plays an important role during dynamic morphogenetic events. Furthermore, the expression of highly sialylated neural cell adhesion molecule by astrocytes and Müller cells in adult optic nerves and retinae suggests some histogenetically plastic functions for these cells in the adult mouse visual system.

Ultra structural Study of the Human Pineal Gland in Aged Patients Including a Centenarian

An ultrastructural study of human pineal glands obtained at autopsy from 7 patients older than 70 years was conducted in order to clarify the functional anatomy of the pineal in the aged. By light microscopy, the pineal glands from aged patients were parenchymatous and almost indistinguishable from those of younger controls. Electron microscopy of the pineal parenchymal cells revealed deep nuclear indentations, synaptic ribbons and ribbon fields, Golgi apparatus, lipofuscin granules and microtubular sheaves in all subjects, cilia with a 9 + 0 pattern in a few, and lamellated structures suggestive of the outer segment of photoreceptor cells very rarely. Microtubules were numerous in the cytoplasmic processes and bulbous endings. Fibrous astrocytes located between the pinealocytes showed long and thin cytoplasmic processes containing numerous glial filaments. Two types of nerve bouton were present in the pineal parenchyma, one of which contained clear vesicles forming synapse-like contacts with pinealocytes. There were no significant age-related changes in these features in a qualitative comparison with pineal glands from 5 adult patients younger than 70 years. These findings indicate that even in advanced age, the human pineal gland maintains some functions, such as intercellular communication and photoreception, in common with the pineal in lower vertebrates.

Translocation of a photoreceptor-specific MEKA protein by light

Light- or dark-adapted retinal sections from bovine, chick and mouse were provided for immunohistochemistry with antiserum against a MEKA protein which is specific to photoreceptor cells. In dark-adapted retinal sections, the anti-MEKA serum heavily stained outer segments of photoreceptor cells and lightly stained inner segments. The reverse staining pattern was observed in light-adapted retinal sections. These results indicate that the translocation of MEKA protein in a photoreceptor cell is regulated by light dark.

Transplantation of retinal pigment epithelial cells to immature and adult rat hosts: Short- and long-term survival characteristics

Retinal pigment epithelial (RPE) cells isolated from 6–8-day-old pigmented Long-Evans rat eyes were successfully grafted onto Bruch's membrane in albino Sprague-Dawley hosts ranging in age from 10 days postnatal to young adulthood. Injections of RPE cells were made into the subretinal space using a lesion paradigm which penetrates through the dorsal surface of the eye cutting through the sclera and choroid. Suspensions of pigmented RPE cells were labeled with Nuclear Yellow prior to transplantation, and at 1 week after grafting, the transplanted RPE cells were found attached to previously denuded areas of Bruch's membrane. The grafted RPE cells were positively identified by double labeling — only those RPE cells with melanosomes in their cytoplasm showed fluorescent Nuclear Yellow labeling; the host albino RPE cells showed no nuclear labeling. The grafted RPE cells developed a normal relationship with photoreceptor cell outer segments as seen by electron microscopy. When compared with intact retinas or host control areas there were no significant differences in the thickness of the outer nuclear layer or in the lengths of photoreceptor inner and outer segments underneath the grafted RPE cells for at least 3 months after transplantation, which was the longest survival time examined.

Ultrastructural visualization of primate cone photoreceptor matrix sheaths.

Glycoconjugates, including glycolipids, glycoproteins, and proteoglycans, are present in the plasma membrane of photoreceptor cells and in the interphotoreceptor matrix surrounding photoreceptor cell ellipsoids and outer segments. Although the precise function of these molecules is unknown, they may be important in mediating photoreceptor-pigment epithelial cell interactions, outer segment membrane assembly, and/or disc shedding. Lectins, affinity ligands for defined carbohydrate sequences, have proven particularly useful in studying the glycoconjugate composition of the interphotoreceptor matrix. The peanut lectin selectively binds to domains of the interphotoreceptor matrix surrounding cone ("cone matrix sheaths"), but not rod inner and outer segments. This is evidence for the existence of chemical and structural heterogeneity within the interphotoreceptor matrix. The studies described herein utilized ultrastructural pre-embedding histochemical labeling to assess whether, in addition to the surrounding interphotoreceptor matrix, peanut lectin binding is associated directly with that plasma membrane of cone inner and outer segments. This study confirms that ferritin-conjugated peanut agglutinin binds to cone matrix sheaths, and, in addition, provides ultrastructural evidence for the presence of binding to the plasma membrane surrounding cone inner and outer segments. The data suggest that cone membrane-associated peanut agglutinin-binding molecules may differ from those located within cone matrix sheaths.

Presence of retina-specific proteins in the lamprey pineal complex

The pineal complex of river lamprey reacted with the antisera raised against retina specific proteins including bovine opsin, chick visinin and frog light-sensitive cyclic GMP phosphodiesterase (PDE). Immunoreactive materials stained with anti-opsin were evenly located at the outer segment of photoreceptor cells in the pineal organ and also found in the parapineal organ. Although anti-visinin stained the pineal and parapineal photoreceptor cells, the immunopositive photoreceptor cells were observed only at the lateral portion and not at the medial portion of the pineal organ. No immunoreactive materials were found in the pineal complex by the anti-PDE, whereas the anti-PDE reacted with photoreceptor cells of the retinal tissue. The data suggest that the pineal and parapineal retinas of lamprey contain opsin- and visinin-like proteins with different distribution in their photoreceptor cell layer as found in the lamprey retinal tissue.

Monoclonal antibodies react with neuronal subpopulations in the human nervous system.

Monoclonal antibody probes were used to identify antigenic cross reactivities among neuronal subpopulations and to dissect the human nervous system at several levels of organization. Six monoclonal antibodies, prepared with immunogens from Drosophila melanogaster or human nervous tissue, were used to localize antigens immunocytochemically in normal adult human neocortex, hippocampus, cerebellum, spinal cord, and retina. Four of the six antibodies were neural specific in their reactivity and each stained a unique combination of neurons. The antibodies reacted with at least three subpopulations of cerebral cortical neurons, including discrete populations of pyramidal and nonpyramidal cells. Components of a widely distributed functional system within the spinal cord and cerebellum were labelled by one antibody, which reacted with neurons in the nucleus dorsalis of Clarke, deep cerebellar nuclei, and Purkinje cells. At the single-cell level, three of the monoclonals differentially labelled the photoreceptor cell outer segment, inner segment, and perikaryon. Three of the six antibodies were reactive with specific protein bands on immunoblots of tissue homogenates. This monoclonal antibody panel provides a novel and potentially useful method of analysis of the organization of the normal and diseased human nervous system.

Light-stimulated protein movement in rod photoreceptor cells of the rat retina

We examined the intracellular distribution of three proteins involved in the cyclic GMP cascade of visual transduction; cGMP phosphodiesterase, the α-subunit of G-protein and arrestin. In adult rats, light-induced changes in the amounts of G and arrestin in the photoreceptor cell outer segments were observed both by polyacrylamide gel analysis of purified ROS and by immunocytochemical localization on retinal sections. In dark conditions, G was concentrated in the outer segments of photoreceptor cells while in the light G α was seen in the inner segments and the outer nuclear layer. Arrestin had the opposite distribution, appearing in the inner segments and outer nuclear layer under dark conditions and in the ROS under light conditions. In contrast, PDE, the enzyme which is activated by G and inhibited by arrestin showed no light-stimulated movement. In both light- and dark-adapted retinas, PDE was localized primarily in the outer segments of the photoreceptor cells.

Transferrin in chick retina: distribution and location during development

Chick retinas from embryonic day 6 (E6) to 3 weeks post-hatching were examined for the presence and location of endogenous transferrin. Immunocytochemistry revealed that transferrin was differentially distributed in retinal layers. Furthermore, the pattern of transferrin distribution changed with developmental age. At day E6, transferrin was found in 2 distinct bands which were located in the area of the Müller cell end-feet. By day E9, additional regions of transferrin immunoreactivity could be found in the inner and outer plexiform layers (IPL, OPL) and the nerve fiber layer (NFL). These latter 3 bands (IPL, OPL and NFL) became more prominent from E9 until E17 as the synaptic layers and nerve fiber layer increased in size, density and maturation. Perikarya in the nuclear layers were negative. At day E17 and later, the newly forming outer segments of photoreceptor cells were strongly reactive for transferrin while the somas of the photoreceptor cells, in the ONL, were negative. Retinas from chicks 1 day to 3 weeks post-hatching retained strong immunoreactivity for transferrin in the photoreceptor cell outer segments and OPL, lessened immunoreactivity in the IPL and loss of immunoreactivity in the NFL. Iron distribution in the retina for all ages examined showed only 2 bands that locally corresponded to the Müller cell end-feet. Iron stores were not found in the synaptic layers or photoreceptor cell outer segments. These studies suggest an iron storage function for retinal glia and a role for transferrin in neuronal development and differentiation.

Monoclonal antibody 18B8 detects gangliosides associated with neuronal differentiation and synapse formation.

Mouse monoclonal antibody 18B8 detects developmentally regulated antigens in chicken retina and brain. The antigens detected by immunofluorescence appear initially on cell bodies in retinas of 6-13 day embryos. In older embryos during synapse formation and in adults, the antigen is localized in discrete laminae within the inner synaptic layer of retina and also is present in the outer synaptic layer and the outer segments of photoreceptor cells. The antigens from retina and brain were purified partially and were shown to be gangliosides of unknown structure that contain at least two sialic acid residues. Gangliosides that are recognized by antibody 18B8 change both qualitatively and quantitatively during neuronal development. These changes were correlated with the spatial and temporal changes in antigen expression detected histochemically.