The left eye was separated using protein extraction solution (iNtRON, Seoul, Korea), and the obtained protein was quantified using a BCA protein assay kit (BIO-RAD, Hercules, CA, USA). Equal amounts of protein were mixed 1:1 with sample buffer (Laemmli sample buffer (BIO-RAD) and 2-mercaptoethanol (BIO-RAD) mixed at 19:1) in each group and then heated at 99°C for 5 minutes. Heated protein was electrophoresed via 12% SDS polyacrylamide gel and transferred by electrophoretic means to nitrocellulose membrane. The membrane was blocked with 5% skim milk at room temperature, and then primary antibodies against the following proteins were used: rhodopsin (Abcam, Burlingame, CA, USA), opsin (Millipore, Burlington, MA, USA), heme oxygenase (HO-1, Cell Signaling, Beverly, MA, USA), nuclear enzyme erythroid 2-related factor-2 (NRF2, Santa Cruz Biotechnology, Santa Cruz, CA, USA), caspase 3 (Cell Signaling), and poly (ADP-ribose) polymerase (PARP, Cell Signaling). Horseradish-peroxidase-conjugated anti-mouse, and anti-rabbit secondary antibody were diluted in TBS-T solution. All western blots were re-probed with anti-β-actin antibody (Cell Signaling) to ensure protein loading. Bands were visualized with EZ-capture ST (ATTD) reagents according to the manufacturer's instructions.
Rhodopsin
Manufactured by Abcam
Sourced in United States, United Kingdom
Rhodopsin is a light-sensitive G-protein-coupled receptor found in the retinal photoreceptor cells of the eye. It plays a central role in the visual transduction process, converting light energy into electrical signals that are transmitted to the brain.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Glutamine synthetase, by Abcam (2 mentions)
α sma, by Abcam (2 mentions)
P smad3, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (2 mentions)
P smad2, by Abcam (2 mentions)
Tyrp1, by Abcam (2 mentions)
Bca protein assay kit, by Bio-Rad (1 mentions)
Poly adp ribose polymerase parp, by Cell Signaling Technology (1 mentions)
Protein extraction solution, by Bio-Rad (1 mentions)
2 mercaptoethanol, by Bio-Rad (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Cell Signaling Technology (1 mentions)
Heme oxygenase 1, by Cell Signaling Technology (1 mentions)
2 nrf2, by Santa Cruz Biotechnology (1 mentions)
Glucagon, by Merck Group (1 mentions)
Gtu 88, by Merck Group (1 mentions)
Gamma tubulin, by Merck Group (1 mentions)
Gm130, by BD (1 mentions)
13 protocols using rhodopsin
1
Protein Expression Analysis in Eye
2
Immunofluorescence Labeling of Diverse Cellular Markers
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Aquaporin-2, rabbit polyclonal (Sigma); Gamma tubulin, mouse monoclonal clone GTU-88 (Sigma); Arl13b, mouse monoclonal clone N295B/66 (NeuroMab, University of California Davis); T1α, hamster monoclonal clone 8.1.1 (Developmental Studies Hybridoma Bank, University of Iowa); Perilipin-A, goat polyclonal (Abcam, Cambridge, MA); Insulin, guinea pig polyclonal (DAKO); Glucagon, mouse monoclonal clone K79bB10 (Sigma); Helix pomatia agglutinin, Alexa488 conjugated (Thermo Fisher Scientific); Lotus Tetragonolobus Agglutinin, fluorescein labeled (Vector Labs, Burlingame, CA); Arf4, rabbit polyclonal antibody against residues 98–114 of mouse Arf4; Rhodopsin, monoclonal clone 1D4 (Abcam); Peripherin, rabbit polyclonal antibody (Gabriel Travis, University of California Los Angeles); Phosducin, mouse monoclonal clone G-7 (Santa Cruz, Dallas, TX); GM130, mouse monoclonal clone 35 (BD Biosciences, San Jose, CA); B-actin, mouse monoclonal clone C4 (Santa Cruz); Hsp90, rabbit polyclonal (Santa Cruz).
3
Immunostaining of Mouse Retinal Cells
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Transduced mouse retinas were dissected and fixed in 4% paraformaldehyde for 30 min at room temperature. The retinas were incubated in PBS with 1% Triton X-100, 0.5% Tween 20 for 1 h at room temperature and in 10% normal goat serum for 1 h at room temperature and then incubated overnight at 4 °C with primary antibody: Brn3a (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rhodopsin (1:500; Abcam, Cambridge, UK), PKCα (1:500; Abcam), calbindin (1:500; Abcam) and glutamine synthetase (GS) (1:500; Abcam) in blocking buffer. Secondary anti-rabbit IgG and anti-mouse IgG conjugated with Alexa TM594 and Alexa TM555, respectively (1:1,000; Molecular Probes), were applied for 1 h at room temperature. The retinas then were flat-mounted and the sections mounted on slide glass.
4
Flow Cytometry Characterization of hPBMCs
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Flow cytometry was performed to quantify the expression of cell-specific markers in both freshly isolated and pre-induced hPBMCs. Both direct and indirect immunolabeling methods were used. Antibodies used for direct labeling (FITC- or PerCP-conjugated) were specific for human CD3, CD16, CD19, or CD45 (Invitrogen, USA), and each staining trail was compared to a specific isotype control. Primary antibodies used for indirect labeling were specific for human nestin (Millipore, USA), vimentin, MAP2, GFAP, synapsin, rhodopsin (all from Abcam, England), or β-tubulin III (Sigma-Aldrich, USA). The secondary antibodies were FITC- conjugated goat anti-rabbit (Cell Signaling Technology, USA) or PE-conjugated goat anti-rabbit (SouthernBiotech, USA). Fix and Cell Permeabilization reagents (Invitrogen, USA) were used for labeling intracellular antigens. Cell suspensions were stained and counted by flow cytometry according to the manufacturer’s directions. Cell suspensions treated with secondary antibodies only were measured as the isotype controls for indirect labeling. After staining, cell suspensions were tested immediately by flow cytometry (Becton, Dickinson Company, USA) using FCS Express V3 software for data analysis. The positive value of the isotype controls ranged from 0% to 1%.
5
Immunofluorescence Analysis of Stem Cell Markers
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For immunofluorescence analysis, fixed cells, cryosections from eyes and matrigel containing transplanted cells in nude mice, were permeabilized with 0.25% Triton X-100 (Sigma) for 2 min, washed with PBS, and then blocked with 2% bovine serum albumin (BSA, Sigma) in PBS. The sections were incubated with the primary antibodies (1:200) against OCT4, NANOG, SOX2, ZO-1, FN1, LIN7A, PARD6B, CRALBP, BEST1, PPM1A, MITF-A, CRX and C-MYC (Proteintech, Rosemont, IL); against SSEA4, TYRP1, α-SMA, FOXF2, MYOSIN, p-SMAD2, p-SMAD3, NR2E1, Rhodopsin, and Ki67 (Abcam, Cambridge, UK); against RPE65 (Novus Biologicals, Centennial, CO), and against FLAG (1:1000, MBL International, Woburn, MD) overnight at 4°C. They were then washed three times with PBS, followed by incubation with the fluorescent secondary antibodies (1:1000, invirogen) overnight. F-actin was stained with phalloidin-iFluor 555 (Yeasen Biotech, Shanghai, China). 4,6 diamidino-2-phenylindole dihydrochloride (DAPI, Sigma) was used to indicate the nucleus. The samples were then examined by fluorescence microscope (Olympus IX73, Tokyo, Japan). Antibodies were listed in key resources table .
6
Retinal Protein Extraction and Western Blot
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The retina protein was extracted using RIPA buffer (P0013B, Beyotime, China) with PMSF and phosphatase inhibitors. The concentration of extracted protein was measured with BCA protein concentration assay kit (P0009, Beyotime, China). According to the measured value, the samples were adjusted to the same protein concentration (0.8 μg/μL) and denatured. 15 μL of each sample containing equal amounts of protein (12 μg) was taken for electrophoresis and transferred. The PVDF membrane with Western blotting was blocked with blocking solution (5% skimmed milk powder prepared with TBST) for 2 h, and then western blotting was incubated with primary antibody mTOR, 1:1,000 (Abcam), p-mTOR, 1:1,000 (Abcam), Rhodopsin, 1:500 (Abcam), and GAPDH, 1:10,000 (Prteintech) overnight at 4°C. After overnight, the membrane was washed three times with TBST and then incubated with goat anti-mouse IgG secondary antibodies 1:10,000 (Proteintech) and goat anti-rabbit IgG secondary antibodies 1:10,000 (Proteintech). After washed three times with TBST, the PVDF membrane was added with the ECL droplet to make it uniformly distribute on the surface of the whole membrane and was put into the gel imaging system to take photos.
7
Immunochemistry analysis of eye tissue
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PSD95, Syt1a and mGluR5 immunochemistry were done on eye cryosections (14 µm), whereas Rhodopsin was tested on paraffin sections (4 µm). Briefly, sections were heated 30 minutes in sodium citrate buffer pH 6.0 and then blocked 2 hours in blocking buffer (10% FBS, 2% BSA, 0.3% Triton X-100 and 0.1% NaN3) at room temperature. Sections were incubated with a 1∶500 dilution of PSD95, Syt1a, mGluR5 or Rhodopsin antibodies (Abcam, Paris, France) overnight at 4°C in blocking buffer. After washing them three times in TBS pH 7.6, sections were stained with 1∶100 dilution goat anti-mouse secondary antibody (Life Technologies, Carlsbad, USA) (FITC for PSD95, Syt1a and mGluR5 and TRITC for Rhodopsin). DAPI at 10 µg/mL (Roche Applied Science, Indianapolis, USA) was then applied on sections for 10 minutes at room temperature. Sections were washed three times in TBS pH 7.6, mounted with Fluoromount (Vector Laboratories, Burlingame, USA) and stored at 4°C. Imaging was realised on a Carl Zeiss apotome (40X, 40/1.3) and analysed on AxioVision software thank to the Cytometry and Imagery platform of the CBM (Orléans, France).
8
Immunofluorescence Analysis of Eye and Cell Samples
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For immunofluorescence analysis, fixed cells, cryosections from eyes and matrigel containing transplanted cells in nude mice, were permeabilized with 0.1% Triton X-100 (Sigma) for 2 min, washed with PBS, and then blocked with 2% bovine serum albumin (BSA, Sigma) in PBS. The sections were incubated with the primary antibodies (1:200) against ZO-1, FN1, CRALBP, and BEST1 (Proteintech, Rosemont, IL, USA); against TYRP1, α-SMA, pSMAD2, pSMAD3, Rhodopsin, PEDF, and Ki67 (Abcam, Cambridge, UK); against pp38 (CST, Danvers, MA, USA) overnight at 4 °C. They were then washed three times with PBS, followed by incubation with the fluorescent secondary antibodies (1:1000, Invitrogen) overnight. 4,6 diamidino-2-phenylindole dihydrochloride (DAPI, Sigma) was used to indicate the nucleus. The samples were then examined by fluorescence microscope (Olympus IX73, Tokyo, Japan). Antibodies were listed in Table S3 .
9
Characterization of Differentiated BMSCs
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BMSCs were fixed with 4 % paraformaldehyde at days 7 or 14 post-neural induction, blocked in 1 % bovine serum albumin and 0.2% Triton X-100 (Sigma-Aldrich, MO, USA), and incubated at 37°C for 1h with primary antibodies against glial fibrillary acidic protein (GFAP; 1:200; rabbit anti mouse; Santa Cruz, CA, USA), nestin (a neural stem cell marker; 1:200; Sigma, goat anti mouse; MO, USA), neuron-specific nuclear protein (NeuN; retinal ganglion cell marker; 1:200; rabbit anti mouse; Abcam, MA, USA), RPE65 (an RPE marker; 1:200; rabbit anti mouse; Santa Cruz, CA, USA) and rhodopsin (a photoreceptor marker; 1:200; rabbit anti mouse; Abcam, MA, USA). The cells were subsequently incubated at 37°C with fluorescein-conjugated secondary antibodies (Dako, CA, USA), mounted and observed under a fluorescence microscope.
10
Immunofluorescent Analysis of Retinal Cell Types
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The printed bioink containing the MIO-M1 cells was fixed with 4% PFA for 30 min at room temperature, followed by permeabilization with 0.1% Triton-X for 7 min. The samples were blocked with 10% normal goat serum (Vector Laboratory, San Francisco, CA, USA) for 1 h and incubated with a 1:100 dilution of blue opsin (Millipore, Burlington, MA, USA), red-green opsin (Millipore), rhodopsin (Abcam), glial fibrillary acidic protein (GFAP; Abcam), and glutamine synthetase (GS, Abcam) antibodies overnight at 4 °C (Table S1 ). After washing with PBS containing Tween-20 (PBST) three times, the samples were incubated with a 1:1000 dilution of secondary antibody conjugated with Alexa Fluor 488 or 594 (Invitrogen, Carlsbad, CA, USA). Then, the samples were washed with PBST three times and co-stained with DAPI for visualizing the nuclei. The fluorescence signal was observed under a fluorescence microscope (Zeiss).
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