Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, M, R||ICC, WB||M||Purified||Monoclonal Antibody|
|Description||Anti-Rhodopsin Antibody, clone RET-P1|
|Presentation||Purified immunoglobulin. Liquid in 10 mM PBS, pH 7.4 with 0.2% BSA and 15 mM sodium azide.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Maintain at 2-8°C in undiluted aliquots for up to 6 months.|
|Material Size||500 µL|
Anti-Rhodopsin Antibody, clone RET-P1 SDS
|MOUSE ANTI-RHODOPSIN MONOCLONAL ANTIBODY - 2121742||2121742|
|MOUSE ANTI-RHODOPSIN - 2492131||2492131|
|MOUSE ANTI-RHODOPSIN - 3184121||3184121|
|MOUSE ANTI-RHODOPSIN - 3188689||3188689|
|MOUSE ANTI-RHODOPSIN - 3219928||3219928|
|MOUSE ANTI-RHODOPSIN - 3288789||3288789|
|MOUSE ANTI-RHODOPSIN - 3380383||3380383|
|MOUSE ANTI-RHODOPSIN - 3458137||3458137|
|MOUSE ANTI-RHODOPSIN - 3483449||3483449|
|Reference overview||Application||Species||Pub Med ID|
|Canine retina has a primate fovea-like bouquet of cone photoreceptors which is affected by inherited macular degenerations.|
Beltran, WA; Cideciyan, AV; Guziewicz, KE; Iwabe, S; Swider, M; Scott, EM; Savina, SV; Ruthel, G; Stefano, F; Zhang, L; Zorger, R; Sumaroka, A; Jacobson, SG; Aguirre, GD
PloS one 9 e90390 2014
Retinal areas of specialization confer vertebrates with the ability to scrutinize corresponding regions of their visual field with greater resolution. A highly specialized area found in haplorhine primates (including humans) is the fovea centralis which is defined by a high density of cone photoreceptors connected individually to interneurons, and retinal ganglion cells (RGCs) that are offset to form a pit lacking retinal capillaries and inner retinal neurons at its center. In dogs, a local increase in RGC density is found in a topographically comparable retinal area defined as the area centralis. While the canine retina is devoid of a foveal pit, no detailed examination of the photoreceptors within the area centralis has been reported. Using both in vivo and ex vivo imaging, we identified a retinal region with a primate fovea-like cone photoreceptor density but without the excavation of the inner retina. Similar anatomical structure observed in rare human subjects has been named fovea-plana. In addition, dogs with mutations in two different genes, that cause macular degeneration in humans, developed earliest disease at the newly-identified canine fovea-like area. Our results challenge the dogma that within the phylogenetic tree of mammals, haplorhine primates with a fovea are the sole lineage in which the retina has a central bouquet of cones. Furthermore, a predilection for naturally-occurring retinal degenerations to alter this cone-enriched area fills the void for a clinically-relevant animal model of human macular degenerations.
|Vibratome sectioning mouse retina to prepare photoreceptor cultures.|
Clérin, E; Yang, Y; Forster, V; Fontaine, V; Sahel, JA; Léveillard, T
Journal of visualized experiments : JoVE 2014
The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 µm thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 µm is discarded before 200 µm of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 °C. Pieces of outer layer are incubated in 500 µl of Ringer's solution with 2 units of activated papain for 20 min at 37 °C. The reaction is stopped by adding 500 µl 10% fetal calf serum (FCS) in Dulbecco's Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 µl of DMEM and seeded at 1 x 10(5) cells/cm(2). The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.
|Structural and functional effects of hemiretinal endodiathermy axotomy in cynomolgus macaques.|
Dashek, RJ; Kim, CB; Rasmussen, CA; Hennes-Beean, EA; Ver Hoeve, JN; Nork, TM
Investigative ophthalmology & visual science 54 3479-92 2013
Outer retinal injury has been well described in glaucoma. To better understand the source of this injury, we wanted to develop a reliable model of partial retinal ganglion cell (RGC) axotomy.Endodiathermy spots were placed along the inferior 180° adjacent to the optic nerve margin in the right eyes of four cynomolgus monkeys. Fluorescein angiography, spectral domain optical coherence tomography (SD-OCT), and multifocal electroretinography (mfERG) were performed at various intervals. Two animals were sacrificed at 3 months. Two animals were sacrificed at 4 months, at which time they underwent an injection of fluorescent microspheres to measure regional choroidal blood flow. Retinal immunohistochemistry for glial fibrillary acidic protein (GFAP), rhodopsin, S-cone opsin, and M/L-cone opsin were performed, as were axon counts of the optic nerves.At 3 months, there was marked thinning of the inferior nerve fiber layer on SD-OCT. The mfERG waveforms were consistent with inner but not outer retinal injury. Greater than 95% reduction in axons was seen in the inferior optic nerves but no secondary degeneration superiorly. There was marked thinning of the nerve fiber and ganglion cell layers in the inferior retinas. However, the photoreceptor histology was similar in the axotomized and nonaxotomized areas. Regional choroidal blood flow was not affected by the axotomy.Unlike experimental glaucoma, hemiretinal endodiathermy axotomy (HEA) of the RGCs produces no apparent anatomic, functional, or blood flow effects on the outer retina and choroid.
|Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa.|
Tucker, BA; Mullins, RF; Streb, LM; Anfinson, K; Eyestone, ME; Kaalberg, E; Riker, MJ; Drack, AV; Braun, TA; Stone, EM
eLife 2 e00824 2013
Next-generation and Sanger sequencing were combined to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP. Induced pluripotent stem cells (iPSCs), generated from the patient's keratinocytes, were differentiated into multi-layer eyecup-like structures with features of human retinal precursor cells. The inner layer of the eyecups contained photoreceptor precursor cells that expressed photoreceptor markers and exhibited axonemes and basal bodies characteristic of outer segments. Analysis of the USH2A transcripts of these cells revealed that one of the patient's mutations causes exonification of intron 40, a translation frameshift and a premature stop codon. Western blotting revealed upregulation of GRP78 and GRP94, suggesting that the patient's other USH2A variant (Arg4192His) causes disease through protein misfolding and ER stress. Transplantation into 4-day-old immunodeficient Crb1 (-/-) mice resulted in the formation of morphologically and immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient act via post-developmental photoreceptor degeneration. DOI:http://dx.doi.org/10.7554/eLife.00824.001.
|Functional and anatomic consequences of subretinal dosing in the cynomolgus macaque.|
Nork, TM; Murphy, CJ; Kim, CB; Ver Hoeve, JN; Rasmussen, CA; Miller, PE; Wabers, HD; Neider, MW; Dubielzig, RR; McCulloh, RJ; Christian, BJ
Archives of ophthalmology (Chicago, Ill. : 1960) 130 65-75 2012
To characterize functional and anatomic sequelae of a bleb induced by subretinal injection.Subretinal injections (100 μL) of balanced salt solution were placed in the superotemporal macula of 1 eye in 3 cynomolgus macaques. Fellow eyes received intravitreal injections (100 μL) of balanced salt solution. Fundus photography, ocular coherence tomography, and multifocal electroretinography were performed before and immediately after injection and again at intervals up to 3 months postinjection. Histopathologic analyses included transmission electron microscopy and immunohistochemistry for glial fibrillary acidic protein, rhodopsin, M/L-cone opsin, and S-cone opsin.Retinas were reattached by 2 days postinjection (seen by ocular coherence tomography). Multifocal electroretinography waveforms were suppressed post-subretinal injection within the subretinal injection bleb and, surprisingly, also in regions far peripheral to this area. Multifocal electroretinography amplitudes were nearly completely recovered by 90 days. The spectral-domain ocular coherence tomography inner segment-outer segment line had decreased reflectivity at 92 days. Glial fibrillary acidic protein and S-cone opsin staining were unaffected. Rhodopsin and M/L-cone opsins were partially displaced into the inner segments. Transmission electron microscopy revealed disorganization of the outer segment rod (but not cone) discs. At all postinjection intervals, eyes with intravitreal injection were similar to baseline.Subretinal injection is a promising route for drug delivery to the eye. Three months post-subretinal injection, retinal function was nearly recovered, although reorganization of the outer segment rod disc remained disrupted. Understanding the functional and anatomic effects of subretinal injection is important for interpretation of the effects of compounds delivered to the subretinal space.Subretinal injection is a new potential route for drug delivery to the eye. Separating drug effects from the procedural effects is critical.
|Localization of acetylcholine-related molecules in the retina: implication of the communication from photoreceptor to retinal pigment epithelium.|
Matsumoto, H; Shibasaki, K; Uchigashima, M; Koizumi, A; Kurachi, M; Moriwaki, Y; Misawa, H; Kawashima, K; Watanabe, M; Kishi, S; Ishizaki, Y
PloS one 7 e42841 2012
It has been long speculated that specific signals are transmitted from photoreceptors to the retinal pigment epithelium (RPE). However, such signals have not been identified. In this study, we examined the retinal expression and localization of acetylcholine-related molecules as putative candidates for these signals. Previous reports revealed that α7 nicotinic acetylcholine receptors (nAChRs) are present in the microvilli of RPE cells that envelope the tips of photoreceptor outer segments (OS). Secreted mammalian leukocyte antigen 6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP-1) is a positive allosteric modulator of the α7 nAChR. Therefore, we first focused on the expression of SLURP-1. SLURP-1 mRNA was expressed in the outer nuclear layer, which is comprised of photoreceptor cell bodies. SLURP-1 immunoreactivity co-localized with rhodopsin and S-opsin in photoreceptor OS, while choline acetyltransferase (ChAT) and high affinity choline transporter (CHT-1) were also expressed in photoreceptor OS. Immunoelectron microscopy identified that the majority of SLURP-1 was localized to the plasma membranes of photoreceptor OS. These results provide evidence that SLURP-1 is synthesized in photoreceptor cell bodies and transported to photoreceptor OS, where SLURP-1 may also be secreted. Our findings suggest that photoreceptor OS communicate via neurotransmitters such as ACh and SLURP-1, while RPE cells might receive these signals through α7 nAChRs in their microvilli.
|Localization of Usher 1 proteins to the photoreceptor calyceal processes, which are absent from mice.|
Sahly, I; Dufour, E; Schietroma, C; Michel, V; Bahloul, A; Perfettini, I; Pepermans, E; Estivalet, A; Carette, D; Aghaie, A; Ebermann, I; Lelli, A; Iribarne, M; Hardelin, JP; Weil, D; Sahel, JA; El-Amraoui, A; Petit, C
The Journal of cell biology 199 381-99 2012
The mechanisms underlying retinal dystrophy in Usher syndrome type I (USH1) remain unknown because mutant mice lacking any of the USH1 proteins-myosin VIIa, harmonin, cadherin-23, protocadherin-15, sans-do not display retinal degeneration. We found here that, in macaque photoreceptor cells, all USH1 proteins colocalized at membrane interfaces (i) between the inner and outer segments in rods and (ii) between the microvillus-like calyceal processes and the outer segment basolateral region in rods and cones. This pattern, conserved in humans and frogs, was mediated by the formation of an USH1 protein network, which was associated with the calyceal processes from the early embryonic stages of outer segment growth onwards. By contrast, mouse photoreceptors lacked calyceal processes and had no USH1 proteins at the inner-outer segment interface. We suggest that USH1 proteins form an adhesion belt around the basolateral region of the photoreceptor outer segment in humans, and that defects in this structure cause the retinal degeneration in USH1 patients.
|Longitudinal assessment of retinal structure and function reveals a rod-cone degeneration in a guinea pig Model initially presented as night blind.|
Racine J, Joly S, Lachapelle P
Documenta ophthalmologica Advances in ophthalmology 2011
We have previously reported a naturally occurring retinopathy in a population of guinea pigs, where the affected animals presented a defect of the rod-mediated vision. The purpose of this study was to investigate if the mutants were affected with a stationary or degenerative retinopathy and to identify the cellular origin of this unique disorder. Electroretinogram (ERG) [postnatal day 1 (P1) to P450], light (LM) and electron microscopy (EM) [P5, P150, P450], and immunohistochemistry [P30, P150, P450] were evaluated from normal and mutant animals. Irrespective of age, the scotopic ERGs of mutants could only be evoked by bright flashes, and the resulting ERGs were of photopic waveform. Interestingly, the amplitude of the cone and the rod/cone a-waves was always of smaller amplitude in mutants, but this difference tended to decrease with age. In contrast, the b-waves were of larger amplitude than normal in photopic ERGs obtained prior to age 25 (days) and prior to age 10 for rod/cone ERGs. LM revealed, in mutants, an absence of the outer segment layer (OSL) with a reduction in the outer nuclear layer (ONL) thickness. EM disclosed the presence of cone outer segment (OS) while no rod OS could be evidenced. Immunohistochemistry revealed the presence of rhodopsin, both cone opsins as well as normal synaptophysin immunoreactivity. Finally, neither the retinal structure nor the function in the mutants achieved normal development. Results suggest that mutant animals are suffering from a degenerative retinal disorder that affects the structure and function of rods and cones.
|Two transcription factors can direct three photoreceptor outcomes from rod precursor cells in mouse retinal development.|
Ng, L; Lu, A; Swaroop, A; Sharlin, DS; Swaroop, A; Forrest, D
The Journal of neuroscience : the official journal of the Society for Neuroscience 31 11118-25 2011
The typical mammalian visual system is based upon three photoreceptor types: rods for dim light vision and two types of cones (M and S) for color vision in daylight. However, the process that generates photoreceptor diversity and the cell type in which diversity arises remain unclear. Mice deleted for thyroid hormone receptor β2 (TRβ2) and neural retina leucine zipper factor (NRL) lack M cones and rods, respectively, but gain S cones. We therefore tested the hypothesis that NRL and TRβ2 direct a common precursor to a rod, M cone, or S cone outcome using Nrl(b2/b2) "knock-in" mice that express TRβ2 instead of NRL from the endogenous Nrl gene. Nrl(b2/b2) mice lacked rods and produced excess M cones in contrast to the excess S cones in Nrl(-/-) mice. Notably, the presence of both factors yielded rods in Nrl(+/b2) mice. The results demonstrate innate plasticity in postmitotic rod precursors that allows these cells to form three functional photoreceptor types in response to NRL or TRβ2. We also detected precursor cells in normal embryonic retina that transiently coexpressed Nrl and TRβ2, suggesting that some precursors may originate in a plastic state. The plasticity of the precursors revealed in Nrl(b2/b2) mice suggests that a two-step transcriptional switch can direct three photoreceptor fates: first, rod versus cone identity dictated by NRL, and second, if NRL fails to act, M versus S cone identity dictated by TRβ2.
|The F-BAR Protein Rapostlin Regulates Dendritic Spine Formation in Hippocampal Neurons.|
Wakita Y, Kakimoto T, Katoh H, Negishi M
The Journal of biological chemistry 286 32672-83. Epub 2011 Jul 15. 2011
Pombe Cdc15 homology proteins, characterized by Fer/CIP4 homology Bin-Amphiphysin-Rvs/extended Fer/CIP4 homology (F-BAR/EFC) domains with membrane invaginating property, play critical roles in a variety of membrane reorganization processes. Among them, Rapostlin/formin-binding protein 17 (FBP17) has attracted increasing attention as a critical coordinator of endocytosis. Here we found that Rapostlin was expressed in the developing rat brain, including the hippocampus, in late developmental stages when accelerated dendritic spine formation and maturation occur. In primary cultured rat hippocampal neurons, knockdown of Rapostlin by shRNA or overexpression of Rapostlin-QQ, an F-BAR domain mutant of Rapostlin that has no ability to induce membrane invagination, led to a significant decrease in spine density. Expression of shRNA-resistant wild-type Rapostlin effectively restored spine density in Rapostlin knockdown neurons, whereas expression of Rapostlin deletion mutants lacking the protein kinase C-related kinase homology region 1 (HR1) or Src homology 3 (SH3) domain did not. In addition, knockdown of Rapostlin or overexpression of Rapostlin-QQ reduced the uptake of transferrin in hippocampal neurons. Knockdown of Rnd2, which binds to the HR1 domain of Rapostlin, also reduced spine density and the transferrin uptake. These results suggest that Rapostlin and Rnd2 cooperatively regulate spine density. Indeed, Rnd2 enhanced the Rapostlin-induced tubular membrane invagination. We conclude that the F-BAR protein Rapostlin, whose activity is regulated by Rnd2, plays a key role in spine formation through the regulation of membrane dynamics.