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.
Ab5407
Manufactured by Merck Group
Sourced in United States
The AB5407 is a laboratory device designed for precision measurement and analysis. It is a compact and versatile instrument that can be used for a variety of applications in research and analytical settings. The core function of the AB5407 is to provide accurate and reliable data through its advanced measurement capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Ab5405, by Merck Group (7 mentions)
Ab15282, by Merck Group (2 mentions)
B 1075, by Vector Laboratories (1 mentions)
Cryostat, by Leica (1 mentions)
C1006, by Beyotime (1 mentions)
Sc 374277, by Santa Cruz Biotechnology (1 mentions)
P0096, by Beyotime (1 mentions)
Sc 8429, by Santa Cruz Biotechnology (1 mentions)
Af6197, by Affinity Biosciences (1 mentions)
Alexa fluor conjugates, by Thermo Fisher Scientific (1 mentions)
Mab386, by Merck Group (1 mentions)
Axio imager, by Zeiss (1 mentions)
Dmi6000 fluorescent microscope, by Leica (1 mentions)
Mab5356, by Merck Group (1 mentions)
S 2000, by Vector Laboratories (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Ab51054, by Abcam (1 mentions)
Ab75285, by Abcam (1 mentions)
Ab7800, by Abcam (1 mentions)
10 protocols using ab5407
1
Immunofluorescence Staining of Retinal Cones
2
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.
3
Retinal Immunohistochemistry and Imaging
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Hematoxylin/eosin staining was performed on retinal sections according to standard techniques. Antibodies used were as follows: 1:200 rabbit anti-S opsin (Millipore AB5407, RRID:AB_177457), 1:250 rabbit anticalcitonin gene-related peptide (CGRP) (Millipore PC205L-100UL, RRID:AB_564312), 1:250 rat antimyelin basin protein (MBP) (Millipore MAB386, RRID:AB_94975); secondary antibodies were Alexa Fluor conjugates from Life Technologies and used at 1:500. FITC-Peanut agglutinin (FITC-PNA) (Sigma L7381) was used at 0.01 mg/ml and Isolectin GS-IB4, Alexa Fluor 647 conjugate (Life Technologies I32450) was used at 1:200. Antibodies were applied in phosphate-buffered saline (PBS) containing 0.5% TritonX-100 and 3% fetal bovine serum and incubated on sections overnight at 4°C. Fluorophore-conjugated proteins were applied to sections during incubation with a secondary antibody; both were diluted in PBS. Retinal sections were imaged at 40 × using a NanoZoomer 2.0HT (Hematoxylin/eosin staining) or a Zeiss Axio Imager (fluorescently labeled sections). Both vibratome sections and cryosections from the brain and spinal cord were imaged using a Leica SP5 confocal. Single confocal slices are shown.
4
Immunohistochemical Analysis of Opsin Expression
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Immunohistochemical (IHC) analysis was performed on 5 µm paraffin embedded sections using standardized lab protocols [19 (link)]. Sections were deparaffinated in a series of xylene and ethanol dips followed by a rinse in PBS. Antigen sites were unmasked by soaking the sections in sodium citrate buffer. Sections were blocked with 2% normal horse serum (S-2000 Vector Labs, CA) in 1× PBS at room temperature for an hour. The primary antibodies rhodopsin (mouse monoclonal, Millipore MAB5356), blue opsin (rabbit polyclonal, Millipore AB5407), and green/red opsin (rabbit polyclonal, Millipore AB5405) were applied at 1:200 dilutions in PBS and incubated at 4 °C overnight. The following day sections were rinsed with PBS and the corresponding secondary antibody was applied in the dark in a 1:400 dilution in PBS: Alexa Fluor 488 goat anti-mouse (Invitrogen A11001) for rhodopsin and Alexa Fluor 488 goat anti-rabbit (Invitrogen A11008) for green and blue opsin slides. These slides were then incubated for an hour in the dark and finally nuclei were labeled with 4,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, dilactate, Invitrogen D3571). Opsin labeling was visualized and imaged using a Leica DMI6000 fluorescent microscope (Leica Microsystems, Wetzlar, Germany) [19 (link)].
5
Localization of Opsin Proteins in Human Skin
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Female human skin was obtained with full written consent adhering to the Declaration of Helsinki principles and under human tissue act guidelines. Facelift or abdominoplasty procedures were always performed in the morning and skin processed in the afternoon of the same day. Seven micrometer cryosections (n ¼ 4 donors, 44-63 years) were fixed in acetone before blocking with 5% bovine serum albumin (BSA) and 5% donkey serum (DKS) in phosphate-buffered saline (PBS) for 1 hour. Primary antibodies were diluted in 1% BSA and 1% DKS and incubated overnight at 48C; 1:200 OPN1-SW (AB5407, Millipore, Amsterdam-Zuidoost, the Netherlands), 1:100 OPN3 (ab66742 for immunohistochemistry (IHC) and ab75285 for immunocytochemistry (ICC), Abcam, Cambridge, UK), 1:200 OPN5 for IHC and 1:500 for ICC (ab199668, Abcam). A negative control (omission of primary antibody) was included in the experimental procedure. Double staining was performed with 1:200 KRT14 (ab51054 or ab7800, Abcam). Incubation with 1:200 Alexa-488 (ab150073, Abcam) and Alexa-647 (ab150115, Abcam) was for 1 hour at 378C. Slides were mounted using VECTASHIELD 1 containing DAPI (VECTOR). Images were taken using a confocal microscope (Leica Microsystems B.V., Amsterdam, the Netherlands).
6
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).
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).
7
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.
8
Immunohistochemical Assay for Opsin Localization
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Immunohistochemistry procedures were performed with polyclonal antibodies raised in rabbits against the last 42 amino acids of the C-terminal of human blue opsin (Sigma-Aldrich; AB5407), against the last 38 amino acids of the C-terminal of human red/green opsins (Sigma-Aldrich; AB5405), or raised in goats against a synthetic peptide with 20 amino acids of human blue opsin (Santa Cruz Biotechnology; sc-14363) (Table 1 ). The specificity of the antibodies for snakes was described previously (Hauzman et al., 2014 (link), 2017 (link); Bittencourt et al., 2019 (link); Gower et al., 2019 (link)). Double labeling with the antibodies against SWS1 and against LWS opsins showed differential labeling of distinct photoreceptor populations (Figure 2 ), further indicating the specificity of both antibodies for particular types of cones. We also assessed the specificity of the two anti-SWS1 antibodies, by incubating 12 μm retinal sections of B. jararaca and C. durissus obtained at −25°C with a cryostat (Leica, CM1100; Nussloch, Germany), with a mixture of both antibodies, rabbit anti-SWS1 (AB5407; 1:200) and goat anti-SWS1 (sc14363; 1:200). Immunofluorescence visualization showed a small number of small single cones labeled by both antibodies (data not shown), indicating the specificity of both antibodies against SWS1 cones.
9
Immunohistochemical Analysis of Mouse Retina
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Eyes were enucleated from euthanized mice, followed by cautery on the limbus to create a mark at the 12 o’clock position and 4% paraformaldehyde fixation. The hematoxylin and eosin stain (H and E) and IHC staining on retinal whole mounts and cryosections were carried out as previously described [55 (link), 57 (link), 58 (link)].
For IHC analysis of cone cells, anti–ARR3 (1:1000, AB15282, Sigma-Aldrich), anti-PNA (1:200, FL-1071, Vectorlabs), anti–Cre Recombinase (1:200, #15,036, Cell Signaling), anti–GlyPh (1:500; kind gift from Dr Pfeiffer-Guglielmi, University of Tübingen, Germany) [31 (link)], anti–M-opsin (1:200, AB5105, Sigma-Aldrich), and anti–S-opsin (1:200, AB5407, Sigma-Aldrich) antibodies were used. The secondary antibody used was Cy™2 AffiniPure Donkey anti-rabbit antibody (dilution, 1:200; Jackson ImmunoResearch). Nuclei were labeled with Hoechst 33,342 (62,249; Thermo Scientific). After being washed three times with PBS, the sections were mounted with fluorescence mounting medium (S3023; Agilent) and viewed using a Nikon confocal microscope (A1RMP, Nikon Corporation, Tokyo, Japan). Image acquisition and processing for three-dimensional rendering was performed using NIS-Elements ver. 4.1 (Nikon).
For IHC analysis of cone cells, anti–ARR3 (1:1000, AB15282, Sigma-Aldrich), anti-PNA (1:200, FL-1071, Vectorlabs), anti–Cre Recombinase (1:200, #15,036, Cell Signaling), anti–GlyPh (1:500; kind gift from Dr Pfeiffer-Guglielmi, University of Tübingen, Germany) [31 (link)], anti–M-opsin (1:200, AB5105, Sigma-Aldrich), and anti–S-opsin (1:200, AB5407, Sigma-Aldrich) antibodies were used. The secondary antibody used was Cy™2 AffiniPure Donkey anti-rabbit antibody (dilution, 1:200; Jackson ImmunoResearch). Nuclei were labeled with Hoechst 33,342 (62,249; Thermo Scientific). After being washed three times with PBS, the sections were mounted with fluorescence mounting medium (S3023; Agilent) and viewed using a Nikon confocal microscope (A1RMP, Nikon Corporation, Tokyo, Japan). Image acquisition and processing for three-dimensional rendering was performed using NIS-Elements ver. 4.1 (Nikon).
10
Immunohistochemical Analysis of Retinal Neurons
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Flat-mounted retinas were fixed with 4% paraformaldehyde (PFA; Electron Microscopy Sciences). Retinas were then embedded in 4% low melting agarose and sectioned into 100 μm thickness with Leica VT1000S vibratome (Leica Biosystems, Deer Park, IL, USA). Retinal sections or flat-mounted retinas were incubated with the primary antibodies for 3 days at 4 °C. The primary antibodies used were rabbit anti-R/G opsin (1:1000, catalog #AB5405, Sigma), rabbit anti-B opsin (1:1000, catalog #AB5407, Sigma, St. Louis, MO, USA), rabbit anti-melanopsin (1:1000, catalog #AB-N38, Advanced Targeting Systems, Carlsbad, CA, USA), chicken anti-GFP (1:1000, catalog #A10262, Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-PKCα (1:600, catalog #P4334, Sigma), mouse anti-Syt2 (1:1000, catalog #Znp-1, DSHB, The University of Iowa, Iowa City, IA, USA), and mouse anti-Nrf1 (1:300, catalog #PCRP-NFR1-3D4; DSHB). Secondary antibodies conjugated with Alexa-488 and -555 (Thermo Fisher Scientific) were used at 1:800 dilution. DAPI (2.5 μg/mL, catalog #D1306; Thermo Fisher Scientific) was used to stain nuclei. Images were captured using a Zeiss LSM 780 confocal microscope (Carl Zeiss, White Plains, NY, USA) and exported as TIFF files into Adobe Photoshop (Adobe Systems, San Jose, CA, USA). Cell counting was conducted using the cell counter plugin in the NIH ImageJ (NIH, Bethesda, MD, USA).
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