Rhodopsin

Manufactured by Merck Group

Sourced in United States

Rhodopsin is a photoreceptor protein found in the retinal cells of the eye. It is responsible for initiating the visual transduction process, which converts light energy into electrical signals that the brain can interpret. Rhodopsin is a member of the G protein-coupled receptor family and is essential for the detection of light in low-light conditions.

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Lab products found in correlation

Calretinin, by Merck Group (3 mentions) Nestin, by Santa Cruz Biotechnology (3 mentions) Glial fibrillary acidic protein (gfap), by Abcam (3 mentions) Streptavidin, by Jackson ImmunoResearch (2 mentions) Lsm 510 exciter microscope, by Zeiss (2 mentions) Hdac4, by Merck Group (2 mentions) Red green opsin, by Merck Group (2 mentions) S opsin, by Merck Group (2 mentions) Cryostat, by Thermo Fisher Scientific (2 mentions) Oct medium, by Sakura Finetek (2 mentions) Protein kinase cα pkcα, by Abcam (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Glutamine synthetase, by BD (1 mentions) Cone arrestin, by Merck Group (1 mentions) P120 catenin, by BD (1 mentions) Plvap, by BD (1 mentions) Ve cadherin, by BD (1 mentions) Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions) Cd11b, by BD (1 mentions) Ab21990, by Abcam (1 mentions)

15 protocols using rhodopsin

Retinal Immunolabeling and Imaging Protocol

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Retinas were fixed in 4% paraformaldehyde in PBS for at least 30min at room temperature for retinal flat mount or cryosection. Primary antibodies: HDAC4 (Sigma, 1:200), Rhodopsin (Rho4D2, 1:100), Red/Green opsin (Millipore, 1:200) and biotin-conjugated Peanut agglutinin (PNA) (1:300). Secondary antibodies: DyLight 594/647-conjugated affiniPure antibodies or streptavidin (Jackson ImmunoResearch). Confocal images were acquired using a Zeiss LSM 510 EXCITER microscope. Images were analyzed in Image J.

Guo X., Wang S.B., Xu H., Ribic A., Mohns E.J., Zhou Y., Zhu X., Biederer T., Crair M.C, & Chen B. (2015). A Short N-terminal Domain of HDAC4 Preserves Photoreceptors and Restores Visual Function in Retinitis Pigmentosa. Nature communications, 6, 8005.

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Immunohistochemical Analysis of Retinal Cell Markers

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The following primary antibodies were used: glutamine synthetase (1:250; BD Biosciences), rhodopsin (1:500; Millipore), cone arrestin (1:500; Millipore), PKCα (1:250; BD Biosciences), MPP4 AK4 (1:200; homemade³⁴ (link)), CRB1 AK2 (pH 1.5) (1:200; homemade⁸ (link)), CRB2 (1:200⁸ (link)), p120-catenin (1:100; BD Biosciences), GFAP (1:200; Dako), CD11b (1:100; BD Biosciences); PLVAP (1:200; BD Pharmingen), and VE-cadherin (1:100; BD Biosciences).

Buck T.M., Vos R.M., Alves C.H, & Wijnholds J. (2020). AAV-CRB2 protects against vision loss in an inducible CRB1 retinitis pigmentosa mouse model. Molecular Therapy. Methods & Clinical Development, 20, 423-441.

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Immunohistochemistry of Xenopus Embryos

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Immunohistochemistry was performed on sections after in situ hybridization using standard procedures. Briefly, the alkaline phosphatase in situ hybridization reaction was stopped with water, followed by PBS-T (PBS pH 7.4, 0.1% BSA (Sigma), and 0.5% Triton X100 (BDH)). Sections were blocked with blocking buffer (PBS-T plus 5% goat serum (Invitrogen) before addition of primary antibody in blocking buffer. The rabbit polyclonal antibodies Prox1 (1:400 dilution; ab37128), Otx2 (1:200; ab21990) (both Abcam), Pax6 (1:100) and Calbindin (1:200; D-28 K) (both Cedarlane), or the mouse monoclonal antibodies Islet1 (1:100; clone 394D5; DSHB, IA, USA), TH (1:100; clone LNC1) and rhodopsin (1:200 dilution; clone Rho 1D4) (both Millipore) were used. Alexa Fluor-tagged secondary antibodies (green and red; 488 and 546 nm emission, respectively; anti-mouse or anti-rabbit) diluted 1:1000 were used to detect the appropriate primary antibody. Of note, it is possible that the Otx2 antibody raised against rabbit OTX2 recognizes both Otx2 and Otx5b in Xenopus[38 (link)].

Bertolesi G.E., Hehr C.L, & McFarlane S. (2014). Wiring the retinal circuits activated by light during early development. Neural Development, 9, 3.

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Retinal Immunolabeling and Imaging Protocol

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Immunohistochemical Characterization of Retinal Progenitor Cells

Cited 2 times

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RPCs were cultured on pure SF, SF:PLCL (1:1), pure PLCL and coverslips (VWR, West Chester, PA) coated with laminin (Sigma-Aldrich, Saint Louis, MO) for 3 days or 7 days fixed in 4% PFA in PBS for 15 min at room temperature. The cells were then washed with PBS 3 times and blocked for 1 hour in a blocking solution (PBS containing 10% (v/v) normal goat serum (Invitrogen), 0.3% Triton X-100 (Sigma-Aldrich) and 0.1% NaN₃ (Sigma-Aldrich))16 (link). The samples were then incubated overnight at 4 °C with the following primary antibodies: Ki-67 (mouse monoclonal, BD, 1:200), rhodopsin (mouse monoclonal Millipore, 1:200), MAP2 (rabbit monoclonal, Epitomics, 1:200) and GFAP (mouse monoclonal, Chemicon, 1:200). Then, samples were washed with PBS and incubated with fluorescent secondary antibodies (Alexa Fluor 546 goat anti-mouse or goat anti-rabbit, 1:300 in PBS, BD) for 1 hour at room temperature. The cell nuclei were counterstained with Slow Fade Gold with DAPI (Invitrogen, Carlsbad, CA)50 (link). Immunostaining-positive cells were detected using a Zeiss LSM710 laser confocal microscope (Carl Zeiss Microscopy, Oberkochen, Germany). The data were collected using Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MD), which includes automated counting software. For each RPC culture, 500–1000 cells were counted in random fields.

Zhang D., Ni N., Chen J., Yao Q., Shen B., Zhang Y., Zhu M., Wang Z., Ruan J., Wang J., Mo X., Shi W., Ji J., Fan X, & Gu P. (2015). Electrospun SF/PLCL nanofibrous membrane: a potential scaffold for retinal progenitor cell proliferation and differentiation. Scientific Reports, 5, 14326.

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Immunohistochemical Staining Protocols

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Antibodies against β-tubulin, actin and acetylated α-tubulin were obtained from Sigma-Aldrich (St. Louis, MO, United States). Peanut agglutinin (PNA) was procured from Vector Labs (Burlingame, CA, United States); Rhodopsin and S-opsin antibodies were purchased from Millipore (Billerica, MA, United States) and SantaCruz, respectively.

Li L., Rao K.N, & Khanna H. (2019). Structural but Not Functional Alterations in Cones in the Absence of the Retinal Disease Protein Retinitis Pigmentosa 2 (RP2) in a Cone-Only Retina. Frontiers in Genetics, 10, 323.

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Western Blot Analysis of Protein Lysates

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Cells were lysed in RIPA buffer containing PMSF and protease inhibitor cocktail (Cat# sc-24948, Santa Cruz) for 30 min on ice and centrifuged for 15 min at 13,000 rpm. Protein concentrations were determined by bicinchoninic acid protein assay kit (Pierce) according to the manufacturer’s instructions. Protein lysates were denatured by adding 2 × laemmli buffer (Cat# 161-0737, Bio-Rad) containing 5% β-mercaptoethanol (Sigma). Protein samples (10 to 30 μg) were separated on 7.5% or 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were blocked in 5% nonfat dry milk in Tris buffered saline plus 0.1% Tween-20 and incubated with primary antibodies overnight at 4°C, followed by 3 PBS washes and incubation with horseradish peroxidase-conjugated secondary antibodies (1:3,000, Santa Cruz) for 1 h at RT. Detection of the immunoreactive bands was performed with the ECL Western Blotting Substrate (Pierce), and chemiluminescence was detected with the Bio-Rad imaging system. PVDF membranes were stripped with glycine buffer plus 0.1% β-mercaptoethanol and re-probed with the appropriate antibodies. Primary antibodies raised against the following proteins were used for western blots: rhodopsin (mouse monoclonal, 1:500; Millipore), Mertk (monoclonal, 1:1,000; abcam), and β-actin (monoclonal, 1:5,000; Sigma).

Tsai Y., Lu B., Bakondi B., Girman S., Sahabian A., Sareen D., Svendsen C.N, & Wang S. (2015). Human iPSC-derived neural progenitors preserve vision in an AMD-like model. Stem cells (Dayton, Ohio), 33(8), 2537-2549.

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Immunohistochemical Analysis of Rat Retinal Slices

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The rat eyes were removed, punctured with a fine gauge needle, and placed in 4% PFA at 4 °C for 12 h. Eyes were then cryoprotected in 30% sucrose for 12 h, embedded in OCT medium (Sakura Finetek). ~10 µm tissue sections were cut using a cryostat (Thermo Scientific) and collected on poly-L-lysine coated slides. Retinal slices were permeabilized with PBS solution containing 0.15% Triton X-100 for 30 min and incubated with 5% bovine serum albumin (BSA) for 1 h to block non-specific antibody binding. Retinal slices were then incubated with the following primary antibodies: TUJ1 (1:300, Abcam), GFAP (1:200, Abcam), GS (1:200, Abcam), Calretinin (1:500, Chemicon), Rhodopsin (1:1,000, Sigma), protein kinase Cα (PKCα, 1:200, Abcam), and Nestin (1:100, Santa Cruz) at 4 °C for 24 h. The slices were washed with PBS and then incubated with the secondary antibody (Invitrogen) overnight at 4 °C. The slides were finally mounted and observed.

Wang J.J., Liu C., Shan K., Liu B.H., Li X.M., Zhang S.J., Zhou R.M., Dong R., Yan B, & Sun X.H. (2018). Circular RNA-ZNF609 regulates retinal neurodegeneration by acting as miR-615 sponge. Theranostics, 8(12), 3408-3415.

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Immunohistochemical Analysis of Rat Retina

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The eyes of SD rats were isolated and the cornea was cut off along the limbus. The cup-shaped retinas were fixed in 4% paraformaldehyde for 12 h, cryoprotected in 30% sucrose for 12 h, and then embedded in optimum cutting temperature (OCT) medium. For immunohistochemistry, retinal slices were incubated in the blocking solution (5% bovine serum albumin, 0.3% Triton X-100 in PBS) for 30 min at 37 °C. Retinal slices were then incubated with the primary antibodies, including TUJ1 (1:300, Abcam), GFAP (1:200, Abcam), GS (1:200, Abcam), calretinin (1:500, Chemicon), rhodopsin (1:1000, Sigma), PKCα (1:200, Abcam), and nestin (1:100, Santa Cruz) at 4 °C for 24 h. The slices were washed with phosphate buffer saline containing 0.1% Tween 20 (PBST) buffer, and then incubated with the fluorescein isothiocyanate (FITC)- or Cy3-conjugated secondary antibody (Invitrogen) overnight at 4 °C. Finally, the slides were observed and imaged.

Wang J.J., Shan K., Liu B.H., Liu C., Zhou R.M., Li X.M., Dong R., Zhang S.J., Zhang S.H., Wu J.H, & Yan B. (2018). Targeting circular RNA-ZRANB1 for therapeutic intervention in retinal neurodegeneration. Cell Death & Disease, 9(5), 540.

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Immunohistochemical Analysis of Retinal Tissues

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The eyes of mice or rats were removed, punctured with a fine gauge needle, and placed in 4% PFA at 4°C for 12 h. Eyes were then cryoprotected in 30% sucrose for 12 h and embedded in OCT medium (Sakura Finetek). Ten‐micrometer tissue sections were cut at −20°C in a cryostat (Thermo Scientific) and collected on the poly‐L‐lysine coated slides. For immunohistochemistry, sections were permeabilized in PBS with 0.2% Triton X‐100 for 20 min and then blocked in PBS with 10% bovine serum albumin (BSA) for 1 h. Retinal sections were incubated with the primary antibodies, including GFAP (1:200, Abcam), GS (1:200, Abcam), NeuN (1:100, Abcam), TUBB3 (1:100, Abcam), calretinin (1:500, Chemicon), calbindin (1:200, Abcam), rhodopsin (1:1,000, Sigma), protein kinase Cα (PKCα, 1:200, Abcam), nestin (1:100, Santa Cruz), or vimentin (1:100, Santa Cruz) at 4°C for 24 h. The sections were washed with PBS and then incubated with FITC‐ or Cy3‐conjugated secondary antibody (1:500, Invitrogen) overnight at 4°C. Slides were finally mounted and observed using an Olympus IX‐73 microscope.

Yao J., Wang X., Li Y., Shan K., Yang H., Wang Y., Yao M., Liu C., Li X., Shen Y., Liu J., Cheng H., Yuan J., Zhang Y., Jiang Q, & Yan B. (2016). Long non‐coding RNA MALAT1 regulates retinal neurodegeneration through CREB signaling. EMBO Molecular Medicine, 8(4), 346-362.

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