Immunofluorescence was performed for both whole mounts and transverse sections of the retina.45 (link) Briefly, retinal cryosections were blocked in 0.5% Triton X-100 and 5% bovine serum albumin in PBS for 1 h at room temperature. Afterward, sections were incubated overnight at 4°C with primary antibodies for anti-IBA1+ (019–19741, 1:1000, Wako, Tokyo, Japan), anti-Rhodopsin (MAB5356, 1:500, Merck, Darmstadt, German), anti-Cone arrestin (AB15282, 1:1000, Merck, Darmstadt, German), and anti-MFRP (AF3445, 1:40, R&D, Minnesota, USA). After washing three times with PBST (1% Tween 20 in PBS), samples were incubated with Alexa 594 (ab150080/ab150116, 1:1000, Abcam, Cambridge, MA, USA) or FITC (PA1-28734, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA) secondary antibodies for 1 h. Subsequently, sections were washed three times with PBS and stained with DAPI. Images were captured using an Olympus FV3000 confocal microscope. The number of infiltrated microglia and GFP density in the RPE layer were quantified by IMARIS software.
Mab5356
Manufactured by Merck Group
Sourced in Germany
MAB5356 is a laboratory instrument manufactured by Merck Group. It is designed for the detection and quantification of specific biomolecules in research and diagnostic applications. The core function of this product is to provide accurate and reliable analytical results.
Automatically generated - may contain errors
Lab products found in correlation
Ab15282, by Merck Group (4 mentions)
Ab5405, by Merck Group (4 mentions)
G3893, by Merck Group (3 mentions)
Tcs sp8 confocal laser scanning microscope, by Leica (3 mentions)
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (3 mentions)
Anti mouse alexa fluor 555, by Thermo Fisher Scientific (3 mentions)
Dmi6000 fluorescent microscope, by Leica (3 mentions)
S 2000, by Vector Laboratories (3 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (3 mentions)
A11008, by Thermo Fisher Scientific (2 mentions)
Ab290, by Abcam (2 mentions)
Ab5407, by Abcam (2 mentions)
Fv3000 confocal microscope, by Olympus (1 mentions)
Ab150080 ab150116, by Abcam (1 mentions)
Af3445, by R&D Systems (1 mentions)
Iblot 2 gel transfer device, by Thermo Fisher Scientific (1 mentions)
Bolt reducing agent, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
β tubulin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 555 647, by Thermo Fisher Scientific (1 mentions)
16 protocols using mab5356
1
Retinal Immunofluorescence Staining Protocol
2
Western Blot Analysis of Rhodopsin
3
Immunostaining Protocol for OXR1, Rhodopsin, and Opsins
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The following antibodies and dilutions were used: OXR1 antibody (1:500) was described previously,35 (link) β-Tubulin (Thermo Fisher; #MA5-16308, 1:2000), Goat Anti-Mouse secondary Ab 680RD (LiCor 926-68070, 1:5000), and Goat Anti-Rabbit Secondary Ab 800CW (Licor 926-32211, 1:5000). For immunostaining studies, the following antibodies and dilutions were used: rhodopsin antibody (Mouse Anti-Rhodopsin monoclonal; MAB5356, 1:500, EMD Millipore), red-green opsin antibody (Rabbit Red/Green opsin; AB5405, 1:500, EMD Millipore), OXR1 antibody, described previously35 (link) (1:200) were used. Secondary antibodies: (Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 546 goat anti-mouse IgG; 1:500; Life Technology Corporation).
4
Immunohistochemistry of Retinal Cell Types
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The primary antibodies/cellular markers used are listed as follows: goat anti-Brn3a (Santa Cruz Biotechnology, 31984, 1:1000), mouse anti-calbindin (Swant, 300, 1:1000), mouse anti-CRALBP (Abcam, ab15051, 1:1000), mouse anti–HPC-1 (Sigma-Aldrich, S0664, 1:500), mouse anti-rhodopsin (Chemicon, MAB5356, 1:500), rhodamine-conjugated peanut agglutinin (PNA; Vector Laboratories, RL-1072-5, 1:500), rabbit anti-melanopsin [Advanced Targeting Systems, AB-N39 (UF008), 1:10,000], rabbit anti-melanopsin (Thermo Fisher Scientific, PA1-780, 1:500), mouse anti–SMI-32 (Covance, SMI32R, 1:1000), and sheep anti-Chx10 (Abcam, ab16141, 1:1000). Alexa Fluor 488/555/647–conjugated fluorescent secondary antibodies raised in donkey were obtained from Thermo Fisher Scientific (USA) or Jackson ImmunoResearch (USA) and used with a final dilution at 1:200.
5
Quantitative Rhodopsin Detection in Ocular Tissue
6
Retinal Cell Labeling and Light-Induced c-Fos Expression
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Mice were anesthetized and perfused intracardially with 0.9% saline, followed by 4% paraformaldehyde in phosphate buffer. Cone and rod photoreceptors were probed with PNA (1:500; RL-1072-5, Vector, USA) and a rhodopsin antibody (1:500; MAB5356, Chemicon, USA), respectively, in retinal sections (20 μm), while ipRGCs and conventional RGCs were stained with a melanopsin antibody [1:10000; AB-N38 (UF008), Advanced Targeting Systems, USA] and a Brn3a antibody (1:1000; 31984, Santa Cruz Biotechnology, USA), respectively, in retinal whole mounts. To quantify light-induced c-Fos expression, after the 25-min light exposure, mice were kept in DD for 90 min and then anesthetized and perfused. c-Fos signals were detected in brain slices (40 μm) or retinal whole mounts with a polyclonal antibody (1:1000; 226 004, Synaptic Systems, Germany). All Alexa Fluor dye–conjugated secondary antibodies were used at a dilution of 1:400.
7
Immunohistochemical Analysis of Retinal Markers
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Immunohistochemical assessment of the retinas was achieved following previously reported methodology52 (link). Briefly, retinal sections were thawed at room temperature, washed 3 times with PB and incubated for 1 h in 0.1 M PB with 10% (v/v) normal donkey serum and 0.5% Triton X-100. After that, sections were immunolabeled overnight at 4 °C under agitation using combinations of primary antibodies at different dilutions in 0.1 M PB with 0.5% Triton X-100: mouse monoclonal anti-rhodopsin (MAB5356, Merk Millipore, Darmstadt, Germany, 1:100), rabbit polyclonal anti-cone arrestin (AB15282, Merk Millipore, 1:200), rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (Iba1) (019-19741, Wako Chemicals, Richmond, VA, USA, 1:1000) and mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (G3893, Sigma-Aldrich, Steinheim, Germany, 1:500). For objective comparison, rd10 and C57BL/6J retinas were processed in parallel. The slides were washed and then incubated with a mixture of corresponding secondary antibodies at a dilution of 1:100 in PB with 0.5% Triton X-100: AlexaFluor 488-anti-rabbit and AlexaFluor 555-anti-mouse (Invitrogen, Carlsbad, CA, USA). When corresponded, the nuclei marker TO-PRO 3-iodide (Invitrogen) was added at a dilution of 1:1000. Images were acquired on a Leica TCS SP8 confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany).
8
Immunohistochemical Analysis of Murine Retinas
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Immunohistochemical assessment of the retinas was achieved following previously reported methodology 52 (link) . Brie y, retinal sections were thawed at room temperature, washed 3 times with PB and incubated for 1 h in 0.1 M PB with 10% (v/v) normal donkey serum and 0.5% Triton X-100. After that, sections were immunolabeled overnight at 4°C under agitation using combinations of primary antibodies at different dilutions in 0.1 M PB with 0.5% Triton X-100: mouse monoclonal anti-rhodopsin (MAB5356, Merk Millipore, Darmstadt, Germany, 1:100), rabbit polyclonal anti-cone arrestin (AB15282, Merk Millipore, 1:200), rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (Iba1) (019-19741, Wako Chemicals, Richmond, VA, USA, 1:1000) and mouse monoclonal anti-glial brillary acidic protein (GFAP) (G3893, Sigma-Aldrich, Steinheim, Germany, 1:500). For objective comparison, rd10 and C57BL/6J retinas were processed in parallel. The slides were washed and then incubated with a mixture of corresponding secondary antibodies at a dilution of 1:100 in PB with 0.5% Triton X-100: AlexaFluor 488anti-rabbit and AlexaFluor 555-anti-mouse (Invitrogen, Carlsbad, CA, USA). When corresponded, the nuclei marker TO-PRO 3-iodide (Invitrogen) was added at a dilution of 1:1000. Images were acquired on a Leica TCS SP8 confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany).
9
Immunohistochemical Profiling of Retinal Cells
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Immunohistochemical assessment of the retinas was achieved following previously reported methodology [52] . Brie y, retinal sections were thawed at room temperature, washed 3 times with PB and incubated for 1 h in 0.1 M PB with 10% (v/v) normal donkey serum and 0.5% Triton X-100. After that, sections were immunolabeled overnight at 4°C under agitation using combinations of primary antibodies at different dilutions in 0.1 M PB with 0.5% Triton X-100: mouse monoclonal anti-rhodopsin (MAB5356, Merk Millipore, Darmstadt, Germany, 1:100), rabbit polyclonal anti-cone arrestin (AB15282, Merk Millipore, 1:200), rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (Iba1) (019-19741, Wako Chemicals, Richmond, VA, USA, 1:1000) and mouse monoclonal anti-glial brillary acidic protein (GFAP) (G3893, Sigma-Aldrich, Steinheim, Germany, 1:500). For objective comparison, rd10 and C57BL/6J retinas were processed in parallel. The slides were washed and then incubated with a mixture of corresponding secondary antibodies at a dilution of 1:100 in PB with 0.5% Triton X-100: AlexaFluor 488anti-rabbit and AlexaFluor 555-anti-mouse (Invitrogen, Carlsbad, CA, USA). When corresponded, the nuclei marker TO-PRO 3-iodide (Invitrogen) was added at a dilution of 1:1000. Images were acquired on a Leica TCS SP8 confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany).
10
Immunohistochemical Staining of Retinal Tissues
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Cryosections were blocked in 10% horse serum, 0.1% Triton X-100 in PBS (PBT), pH 7.5, for 1 h before adding primary antibodies diluted in blocking solution. The following primary antibodies were used (anti-): pAktSer473 (1:500; Cell Signaling #4060), pS6Ser235/236 (1:500; Cell Signaling #4856), Brn3b (1:500; Chemicon #AB5945), Chx10 (1:200; Santa Cruz #sc-21690), cone-arrestin (1:500; Millipore #AB15282), Pax6 (1:500; Covance Research #PRB-278P), Pten (1:500; Cell Signaling #9559), rhodopsin (1:500; Chemicon #MAB5356), calbindin (1:500; Sigma #C9848), GFAP (1:500; Sigma #G9269), CRALBP (1:500; Abcam #ab15051), glutamine synthetase (1:500; Abcam #ab73593), fibronectin (1:200; Abcam #ab2413), laminin (1:500; Sigma #L9393), RPE65 (1:500; ORIGENE #TA309839), Otx2 (1:500; Abcam #ab21990), N-cadherin (1:200; BD Transduction Labs #610920), ZO-1 (1:100; ThermoFisher Scientific #33-9100) and Sox9 (1:500; Millipore #AB5535). Slides were incubated in primary antibodies overnight at 4°C. The next day, slides were washed three times in PBT before incubating with secondary antibodies conjugated with Alexa Fluor 568 (1:500; Molecular Probes) or Alexa Fluor 488 (1:500; Molecular Probes) for 1 h. Slides were then washed three times in PBT before labelling nuclei with DAPI.
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