Ab5405

Manufactured by Merck Group

Sourced in United States

The AB5405 is a laboratory instrument designed for performing various analytical and research tasks. It is a versatile piece of equipment that can be utilized in a wide range of scientific applications. The core function of the AB5405 is to facilitate precise measurements and data collection, but a detailed description of its intended use is not available.

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Lab products found in correlation

Ab5407, by Merck Group (7 mentions) Mab5356, by Merck Group (4 mentions) Lsm 780 confocal microscope, by Zeiss (4 mentions) S 2000, by Vector Laboratories (3 mentions) Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (3 mentions) Ab290, by Abcam (2 mentions) Ab5407, by Abcam (2 mentions) Dmi6000 fluorescent microscope, by Leica camera (2 mentions) A11008, by Thermo Fisher Scientific (2 mentions) Cryostat, by Leica camera (2 mentions) Lsm 710, by Zeiss (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) B 1075, by Vector Laboratories (1 mentions) Ab41672, by Abcam (1 mentions) To pro 3, by Thermo Fisher Scientific (1 mentions) Tcs sp8, by Leica camera (1 mentions) Sc 374277, by Santa Cruz Biotechnology (1 mentions) P0096, by Beyotime (1 mentions) C1006, by Beyotime (1 mentions) Sc 8429, by Santa Cruz Biotechnology (1 mentions)

24 protocols using ab5405

Immunofluorescence Staining of Retinal Cones

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Immunofluorescence staining with frozen sections was performed as previously described [23 (link)]. Frozen retinal sections were rinsed twice in 0.01 M PBS, permeabilized for 30 min in 0.3% TritonX-100, and blocked for 2 h in 5% BSA at room temperature. Then, frozen sections were incubated overnight at 4°C with a rabbit polyclonal anti-human red and green (L)-cone-opsin or blue (S)-cone-opsin antibody (AB5405, AB5407; Millipore, MA, USA) and FITC-conjugated peanut agglutinin (PNA) (B-1075, Vector Laboratories, Burlingame, CA, USA) that was diluted 1 : 400 in 1% BSA and 0.1% TritonX-100. After rinsing with 0.01 M PBS, the retinas were incubated for 1 h in a dark room with goat anti-rabbit IgG that was diluted 1 : 800 in 0.01 M PBS, followed by five rapid rinses with 0.01 M PBS. Then, the sections were incubated for 2 min in 4,6-diamidino-2-phenylindole (DAPI, dilution 1 : 500), rapidly rinsed five times with 0.01 M PBS, and photographed after the addition of antifluorescent quencher.

Zhang H., Li X., Dai X., Han J., Zhang Y., Qi Y., He Y., Liu Y., Chang B, & Pang J.J. (2017). The Degeneration and Apoptosis Patterns of Cone Photoreceptors in rd11 Mice. Journal of Ophthalmology, 2017, 9721362.

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Immunolabeling of Retinal Sections

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After enucleation, eyes were fixed by overnight immersion in 4% paraformaldehyde in PBS at 4°C. Eyes were quenched with 50 mM NH₄Cl for 30 min and then infused successively with 10% and 20% sucrose in PBS, and finally Tissue-Tek “4583” (Miles Inc., Elkhart, IN). Cryosections (8 μm) were cut on a cryostat HM 505E (Microm, Walldorf, Germany) equipped with a CryoJane Tape-Transfer system (Leica Inc., Buffalo Grove, IL). For labeling, sections were washed to remove embedding medium, blocked in PBS supplemented with 1% BSA (PBS/BSA) for 30 min, and incubated with primary followed by secondary antibodies coupled to Alexa 488 as well as with TO-PRO-3 for nuclear labeling (LifeTechnologies, Grand Island, NY) as previously described [10 (link)]. A series of 0.3-μm xy (en face) sections were collected using a laser scanning confocal microscope (Leica TCS-SP8, Exton, PA) using the same acquisition parameters for each channel in the Leica confocal software LAS AF. Antibodies used included rhodopsin (clone B630N, from Dr. G. Adamus, Oregon Health and Science University, Portland, OR, 1:100), red/green opsin (AB5405, Millipore, Billerica, MA, 1:600) and ezrin (ab41672, Abcam, 1:250).

Bonilha V.L., Bell B.A., Rayborn M.E., Samuels I.S., King A., Hollyfield J.G., Xie C, & Cai H. (2017). Absence of DJ-1 causes age-related retinal abnormalities in association with increased oxidative stress. Free radical biology & medicine, 104, 226-237.

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Immunohistochemical analysis of human fetal eyes

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Human fetal eyes were fixed overnight in 4% paraformaldehyde. The samples were then washed in PBS, dehydrated overnight at 4 °C with 20% sucrose solution, embedded in 30% sucrose and OCT (Tissue‐Tek O. C. T. Compound) at a 1:1 ratio, and snap‐frozen in dry ice. Thin (8–10 µm) cryosections were obtained using a cryostat (Leica). For immunohistochemistry, sections were permeabilized for 20 min at 20–28 °C in 0.3% Triton X‐100 (P0096, Beyotime), washed three times in PBS, blocked for 2 h at 37 °C with 10% normal goat serum, and incubated for 16 h at 4 °C with primary antibodies diluted in prefabricated solution. Primary antibodies specific for NRL (1:200; sc‐374277, Santa Cruz), RGR (1:500; ABP56042, Abbkine), CALBINDIN (1:200; 66394‐1‐Ig, Proteintech), PKC (1:200; AF6197, Affinity), CALRETININ (1:200; 92635T, CST), BRN3A (1:200; sc‐8429, Santa Cruz), OPSIN‐Blue (1:300; AB5407, Millipore), OPSIN‐Red/Green (1:300; AB5405, Millipore), and REST (1:200; sc‐374277, Santa Cruz) were used. Subsequently, sections were washed with PBS and incubated for 2 h at room temperature between 20 and 25 °C with secondary antibodies (1:500; A0423/A0521, Beyotime). DAPI (C1006, Beyotime) staining was used to visualize the nuclei. Images were captured using a fluorescence microscope and processed using the ImageJ software.

Li R., Liu J., Yi P., Yang X., Chen J., Zhao C., Liao X., Wang X., Xu Z., Lu H., Li H., Zhang Z., Liu X., Xiang J., Hu K., Qi H., Yu J., Yang P, & Hou S. (2023). Integrative Single‐Cell Transcriptomics and Epigenomics Mapping of the Fetal Retina Developmental Dynamics. Advanced Science, 10(16), 2206623.

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Immunohistochemistry of Cone and Rod Cells

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661 W cells were fixed in 4% paraformaldehyde (PFA) for 15 minutes. Cells were permeabilized and blocked in PBS containing 4% BSA and 0.5% Triton X-100 for 1 hour at room temperature, incubated overnight with primary antibody at 4 °C, and then subjected to immunohistochemistry as previously described36 (link).
Primary antibodies were rabbit anti-opsin blue (1:500; chemicon, AB5407), rabbit anti-opsin red/green (1:500; chemicon, AB5405), rabbit anti-cone arrestin (1:500; Millipore, AB15282) and mouse anti-rhodopsin (1:10000; sigma, o4886).

Lv J.N., Zhou G.H., Chen X., Chen H., Wu K.C., Xiang L., Lei X.L., Zhang X., Wu R.H, & Jin Z.B. (2017). Targeted RP9 ablation and mutagenesis in mouse photoreceptor cells by CRISPR-Cas9. Scientific Reports, 7, 43062.

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Molecular Marker Analysis of Mutants

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Mouse antibody to PKCθ (IgG2a; #610090; BD Biosciences; San Jose, CA, USA), rabbit ZO-1 antibody (61-7300; Invitrogen, Carlsbad, CA, USA), mouse β-catenin antibody (IgG1; #sc-7963; Santa Cruz Biotech; Dallas, TX, USA), mouse antibody to ezrin (E8897; Sigma-Aldrich Corp., St. Louis, MO, USA), goat antibody to ezrin (sc-6409; Santa Cruz Biotech), rabbit antibody to glial fibrillary acidic protein (GFAP; DAKO, Carpinteria, CA, USA), mouse antibody to moesin/radixin (ab50007; Abcam, Cambridge, MA, USA), rabbit antibody to radixin (ab52495; Abcam), mouse antibody to rhodopsin (MS-1233-R7; Thermo Scientific, Waltham, MA, USA), rabbit antibody to blue opsin (AB5407; Chemicon International, Billerica, MA, USA), rabbit antibody to red/green opsin (AB5405; Millipore, Billerica, MA, USA), mouse antibody to E-cadherin (610181; BD Transduction, San Jose, CA, USA), rabbit antibody to pan-cadherin (ab6529-200; Abcam), rabbit antibody to α1 catenin (2028-1; Epitomics, Burlingame, CA, USA), rabbit phospho-ezrin/radixin/moesin (ERM) antibody (#3149; Cell Signaling, Danvers, MA, USA), and rabbit occludin antibody (#71-1500; Invitrogen) were used for marker analyses of the mutant. The loading control sampler kit (#4670; Cell Signaling) included horseradish peroxidase (HRP)-conjugated β-tubulin, β-actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies.

Ji X., Liu Y., Hurd R., Wang J., Fitzmaurice B., Nishina P.M, & Chang B. (2016). Retinal Pigment Epithelium Atrophy 1 (rpea1): A New Mouse Model With Retinal Detachment Caused by a Disruption of Protein Kinase C, θ. Investigative Ophthalmology & Visual Science, 57(3), 877-888.

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Retinal Cell Type Quantification

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Immunohistochemistry analysis was performed on 10μm paraffin embedded serial sections from the enucleated mouse eyes as described in our previous studies⁷². At minimum 100μm of retina/sample was evaluated by IHC. Briefly, sections were blocked with 2% normal horse serum (#S-2000 VectorLabs, CA) in PBS, and incubated with the following cell type-specific primary antibodies in a 1:200 dilution: rhodopsin (mouse monoclonal, Millipore MAB5356); green/red opsin (rabbit polyclonal, Millipore AB5405); blue opsin (rabbit polyclonal, Millipore AB5407; GFP (1:500, rabbit polyclonal, Abcam ab290). The following day, sections were rinsed with PBS and incubated with the corresponding secondary antibody (1:400 Alexa fluor 488 goat anti-rabbit, Invitrogen A11008) and nuclei were stained with 4,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI). Over 500μm of sections/animal were visualized and representative images of IHC labeling were captured using a Leica DMI6000 fluorescent microscope equipped with the appropriate bandpass filter for each fluorochrome. Cell counts were performed in a double-blinded manner over 100μm retinal area. N=10/strain/experimental group.

Li S., Datta S., Brabbit E., Love Z., Woytowicz V., Flattery K., Capri J., Yao K., Wu S., Imboden M., Upadhyay A., Arumugham R., Thoreson W.B., DeAngelis M.M, & Haider N.B. (2020). Nr2e3 is a genetic modifier that rescues retinal degeneration and promotes homeostasis in multiple models of retinitis pigmentosa. Gene Therapy, 28(5), 223-241.

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Quantifying Opsin Expression in RPE

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For the analysis of opsin-positive area, formalin-fixed paraffin-embedded samples were prepared at day 42 post injection (n=4). Cross-section samples were immunostained with anti-HA antibody (Roche, 3F10, 1:1,000), anti-opsin antibody (Millipore, AB5405, 1:1,000), and Alexa Fluor 488 or 594 antibodies (Thermo Fisher Scientific, 1:500). The opsin-positive area corresponding to RPE cells expressing HA-tagged CjCas9 was measured using Image J software (1.47v, NIH) by blinded observers. For the distribution of CjCas9 and eGFP, the eyes were fixed in 4% paraformaldehyde for 1 h at room temperature. RPE complexes (RPE/choroid/sclera) were prepared for immunostaining and then incubated with anti-GFP antibody (Abcam, ab6556, 1:100) overnight at 4 °C. After stain with Alexa Fluor 488 antibodies (1:500), the RPE flat-mounts was imaged using a confocal microscope (LSM 710, Carl Zeiss). The scanning parameters were as follows: scaling (x=0.042 μm per pixel, y=0.042 μm per pixel, z=0.603 μm per pixel), dimensions (x=1,024, y=1,024, channels: 2, 8-bit) with objective C-Apochromat × 40 per 1.20 W Korr M27. ZEN 2 software was used to process the images.

Kim E., Koo T., Park S.W., Kim D., Kim K., Cho H.Y., Song D.W., Lee K.J., Jung M.H., Kim S., Kim J.H., Kim J.H, & Kim J.S. (2017). In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nature Communications, 8, 14500.

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Immunolabeling of Retinal Flat Mounts

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Dissected retinas were fixed in 4% PFA for 90 min at room temperature (RT), washed in PBS several times, permeabilized for 30 min using 1% Triton X-100 and blocked for 1 hr at RT with PBS containing 3% BSA and 0.03% Triton X-100. Retinas for flatmount preparation were incubated with primary antibodies [(Goat anti S Opsin, sc-14363, Santa Cruz Biotechnology, Dallas, TX, USA) and (rabbit anti M opsin, AB5405, Millipore, Billerica, MA, USA)] (1:100) for 4 days at 4°C. For cryosectioing, dorso-ventral orientation of mouse eye was determined using guidelines described previously (Wagner et al., 2000 (link)). Details about antibodies and immunohistochemistry procedure is described in supplemental experimental procedures.

Sawant O.B., Horton A.M., Zucaro O.F., Chan R., Bonilha V.L., Samuels I.S, & Rao S. (2017). The circadian clock gene Bmal1 controls thyroid hormone-mediated spectral identity and cone photoreceptor function. Cell reports, 21(3), 692-706.

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Retinal Cell Type Quantification

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Immunohistochemistry analysis was performed on 10 µm paraffin embedded serial sections from the enucleated mouse eyes as described in our previous studies [72 ]. At minimum 100 µm of retina/sample was evaluated by IHC. Briefly, sections were blocked with 2% normal horse serum (#S-2000 VectorLabs, CA) in PBS, and incubated with the following cell type-specific primary antibodies in a 1:200 dilution: rhodopsin (mouse monoclonal, Millipore MAB5356); green/red opsin (rabbit polyclonal, Millipore AB5405); blue opsin (rabbit polyclonal, Millipore AB5407); GFP (1:500, rabbit polyclonal, Abcam ab290). The following day, sections were rinsed with PBS and incubated with the corresponding secondary antibody (1:400 Alexa fluor 488 goat antirabbit, Invitrogen A11008) and nuclei were stained with 4,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI). Over 500 μm of sections/animal were visualized and representative images of IHC labeling were captured using a Leica DMI6000 fluorescent microscope equipped with the appropriate bandpass filter for each fluorochrome. Cell counts were performed in a double-blinded manner over 100 μm retinal area (N = 10/strain/experimental group).

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Immunostaining of Retinal Rhodopsin and Opsin

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For immunostaining, eyes were fixed in 4% PFA at 4 °C for overnight, cryopreserved in 30% sucrose, and embedded in optimal cutting temperature compound. Sections (14 μm) were collected on a cryostat, dried at RT for 30 min, and fixed in 2% PFA for 10 min. After rinsing, the sections were permeabilized with 0.2% Triton-X-100 for 10 minutes and blocked with 10% goat serum for 1 hour at RT. Anti-Rhodopsin (milipore MAB5316) and anti-Opsin (milipore AB5405) were used as primary antibodies, and goat anti-mouse IgG alexa Fluor® 488 (life technologies, A11029) and goat anti-rabbit IgG alexa Fluor® 594 (life technologies, A11037) were used as secondary antibodies. Each staining was performed on slides from at least three animals per condition. Immunostaining results were observed and photographed using a Zeiss confocal microscope.

Wen B., Li S., Li H., Chen Y., Ma X., Wang J., Lu F., Qu J, & Hou L. (2016). Microphthalmia-associated transcription factor regulates the visual cycle genes Rlbp1 and Rdh5 in the retinal pigment epithelium. Scientific Reports, 6, 21208.

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