The primary antibodies listed in key resources table were used for histochemical staining.66 (link) Mice were fixed by perfusion with 4% paraformaldehyde solution, and the fixed brains were cryo-sectioned into 10-mm-thick coronal sections at the frontal cortex level. After incubation with primary antibodies, the sections were incubated with DyLight 488, DyLight 549, and/or DyLight 649-labeled secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Hoechst 33342 (Sigma-Aldrich) was used for nuclear staining. Imaging was performed using a TiE-A1R confocal microscope (Nikon, Tokyo, Japan) with a 100× objective lens (Plan Apo, NA: 1.45, Nikon). Images were analyzed using IMARIS ver. 9.8.2 (Oxford Instruments, UK).67 (link)
Dylight 549
Manufactured by Jackson ImmunoResearch
Sourced in United States, Panama, France
DyLight 549 is a fluorescent dye provided by Jackson ImmunoResearch. It is designed for use in biological and immunological applications that require a fluorescent label.
Automatically generated - may contain errors
Lab products found in correlation
Dylight 488, by Jackson ImmunoResearch (18 mentions)
Dylight 649, by Jackson ImmunoResearch (9 mentions)
Alexa 488, by Thermo Fisher Scientific (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions)
Fluormount g, by Southern Biotech (4 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Alexa 647, by Thermo Fisher Scientific (3 mentions)
Axioplan 2 microscope, by Zeiss (3 mentions)
Plan neofluor 403 0.75 air objective, by Hamamatsu Photonics (3 mentions)
Charge coupled device camera, by Hamamatsu Photonics (3 mentions)
Fluorescein isothiocyanate (fitc), by Jackson ImmunoResearch (3 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Chicken anti gfp, by Aves Labs (2 mentions)
Citrate buffer based antigen retrieval medium, by Biocare Medical (2 mentions)
Rabbit anti gabaa receptor gamma 2 subunit, by Synaptic Systems (2 mentions)
Mouse anti glyr, by Synaptic Systems (2 mentions)
Guinea pig anti viaat, by Synaptic Systems (2 mentions)
Decloaking chamber, by Biocare Medical (2 mentions)
Anti bik antibodies, by Cell Signaling Technology (2 mentions)
41 protocols using dylight 549
1
Immunohistochemical Staining of Mouse Brain
2
Immunostaining of Neuronal Markers in Tissue Sections
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Sections were cut at a thickness of 7 μm. After removing paraffin, sections were processed in a decloaking chamber (Biocare Medical, Concord, CA) using a citrate-buffer-based antigen retrieval medium (Biocare Medical) for 20 min at 110–115°C. They were then processed in PBS with 15% methanol and 0.3% H2O2 to block endogenous peroxidase activity. Aldehyde groups were removed by incubating the sections in sodium borohydride (1%) in PBS. After these treatments, the slices were incubated in a blocking PBS-based solution containing cold-water fish-skin gelatin (0.1%) and 0.1% Triton X-100. Tissues were then incubated overnight at 4°C with the following primary antibodies: chicken anti-GFP (1:1000 Aves labs, Tigard, OR), rabbit anti-GABAa receptor gamma 2 subunit (1:1500; Synaptic Systems, 224 003, Göttingen, Germany) mouse anti-GlyR (1:1000; Synaptic Systems, 146 011), guinea pig anti-VIAAT (1:1500; Synaptic Systems, data not shown). Primary antibodies were revealed by incubation for 2 h with secondary antibodies coupled to either Alexa Fluor-488 (Invitrogen, Saint Aubin, France) or DyLight 488, DyLight 549, and DyLight 649 (Jackson ImmunoResearch, Newmarket, UK). Sections were finally mounted using Prolong Gold Antifade Reagent (Invitrogen). For all experiments, control sections were incubated without primary antibodies.
3
Immunolabeling of Wnt1-Derived Cells
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Sections were immunolabeled as previously described (Ellisor et al., 2009 (link)). Sagittal sections from Wnt1-Venus embryos at E8.5, E10.5, and E12.5 were analyzed using an anti-GFP antibody (1:600, Molecular Probes, Cat # A-6455). Adult coronal sections for fate mapping experiments were immunolabeled with an anti-β-galactosidase (β-gal) antibody (1:500, Biogenesis, Cat # 4600-1409 or 1:500, Abcam, Catalog # ab9361-250) to identify Wnt1-derived cells (Ellisor et al., 2009 (link)). In addition, anti-Calretinin (1:5000, Chemicon; Billerica, MA; Catalog # AB1550), anti-Calbindin (1:1000, Swant, Catalog # CB3a), and anti-Parvalbumin (1:1000, Sigma, Catalog # P3088-.2ML) antibodies were used as biomarkers. Secondary antibodies were prepared at 1:500 and include: Alexa 488 (Invitrogen; Cat # A-21206, donkey anti-rabbit IgG; Cat # A-21202, donkey anti-mouse IgG; Cat #A-11055 donkey anti-goat IgG), Dylight 549 (Jackson ImmunoResearch Laboratories; Cat #703-505-155, donkey anti-chicken).
4
Immunohistochemistry of Zebrafish Larvae
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Immunohistochemistry was performed as previously described [24] (link) with slight modifications of the fixation times. Zebrafish larvae were fixed with 4% paraformaldehyde/4% sucrose in phosphate buffer with 0.2 mM CaCl2 for 4 h (3 dpf) or 6 h (5 dpf) at 4°C. Following rinse, larvae were permeabilized with ice cold acetone and blocked with phosphate buffered saline (PBS) containing 2% goat serum, 1% bovine serum albumin (BSA), and 1% dimethyl sulfoxide (DMSO). Then they were incubated with primary antibodies diluted in PBS buffer containing 1% BSA and 1% DMSO overnight at 4°C, followed by diluted secondary antibodies coupled to Alexa 488, Alexa 647 (Molecular Probes, Invitrogen), or DyLight 549 (Jackson ImmunoResearch).
5
Comprehensive Immunofluorescence Staining Protocol
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The following primary antibodies were used: anti-BrdU (rat; Abcam, ab6326; 1:200), anti-DsRed (rabbit; Clontech, 632496; 1:500), anti-eGFP (chicken; Life Technologies, A10262; 1:500), anti-GS (mouse; Chemicon, MAB302; 1:500), anti-Otx2 (goat; R&D systems, AF1979; 1:200), anti-pAkt (rabbit; Cell Signaling, 4060; 1:200), anti-Pcna (mouse; Millipore, CBL407; 1:100), anti-pIgf1r (rabbit; Abcam, ab39398; 1:100), anti-Rx2 (rabbit; Reinhardt et al., 2015 (link); 1:500) and anti-Zpr-1 (mouse; ZIRC, ZDB-ATB-081002-43; 1:200). The following secondary antibodies were used (at 1:750): anti-chicken Alexa Fluor 488 (donkey; Jackson, 703-485-155), anti-goat Alexa 633 (donkey; Life Technologies, A-21082), anti-mouse Alexa 546 (goat; Life Technologies, A-11030), anti-mouse Alexa 647 (donkey; Jackson, 715-605-151), anti-rabbit Alexa Fluor 488 (goat; Life Technologies, A-11034), anti-rabbit DyLight549 (goat; Jackson, 112-505-144), anti-rabbit Alexa Fluor 647 (goat; Life Technologies, A-21245) and anti-rat DyLight488 (goat; Jackson, 112-485-143). DAPI (Sigma-Aldrich, D9564) nuclear counterstaining was performed as described by Inoue and Wittbrodt (2011) (link).
6
Immunostaining of Bik in Lung Tissue
7
Immunofluorescent Analysis of Hair Cell Markers in 5-aza Treated Mouse Utricle Cells
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After 5 days of 5-aza treatment, control and treated MUCs were fixed in 4% paraformaldehyde for 10 min at room temperature, followed by incubation in PBS containing 5% donkey serum (Jackson Immunoresearch), and 0.2% Triton X-100 (Sigma) for 20 min. Primary antibodies included epithelial markers anti-E-cadherin (1:200; Santa Cruz) and anti-pan-cytokeratin 26 (PCK26; 1:100; Sigma), and hair cell markers anti-Myosin VI (Myo6; 1:200; Sigma), anti-Myosin VIIa (Myo7a; 1:200; DSHB), anti-Math1 (Atoh1; 1:200; DSHB), anti-Pou4f3 (1:200; Sigma), anti-Espin (1:100; a gift from Dr. James Richard Bartles, Northwestern University), anti-DNMT1 (1:100; Epigentek) and anti-PCNA (1:100; Abcam). Secondary antibodies were Dylight-488-, Dylight-549,- and Dylight-649-conjugated antibodies, with a universal nuclear marker, 4′,6-diamidino-2-phenylindole (DAPI, all from Jackson Immunoresearch). MUC samples were incubated with primary antibodies overnight at 4°C, followed by secondary antibodies incubation for 2–3 h at room temperature. MUCs were observed using a Leica epifluorescence microscope and images were captured using a QImaging monochrome cool CCD camera.
8
Immunohistochemical Analysis of CTGF and p16 in Lung Tissue
9
Immunolabeling Saccular Sensory Epithelia
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Briefly, fish heads were fixed with 4% paraformaldehyde at 4 °C overnight. After rinsing the tissue three times with 0.25% PBST, the sensory epithelia of the saccules were dissected according to a previous report48 (link). For HC counting, the epithelia of the saccules were incubated for 15 minutes in Alexa Fluor 488 Phalloidin (Invitrogen) at 4 °C. For Ribeye b immunofluorescence labelling, the epithelia of the saccules were permeabilized using 1% Triton X-100 at room temperature for 2 hours. Then, the samples were blocked with 10% goat serum in PBS for 1 hour, followed by incubation in the primary antibody (mouse anti-zebrafish Ribeye b monoclonal antibody, kindly provided by Dr. T. Nicolson, Oregon Health & Science University) at 4 °C for 18 hours. After the tissue was rinsed with 0.25% PBST for 2 hours, the samples were stained with a secondary antibody coupled to DyLight 549 (Jackson ImmunoResearch) for 2 hours at room temperature. Finally, the samples were stained with DAPI (Invitrogen).
10
Immunostaining of Primary Polyps
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Samples were anesthetized (primary polyps) and fixed in 4% PFA in PBT for 6 h, washed in PBT and blocked in blocking buffer (80% PBT, 20% sheep serum, 1% bovine serum albuminum) for 2 h at room temperature. They were then incubated in primary antibody (rat, anti-RFP; Chromotek) 1:400 in blocking buffer overnight at 4°C, subsequently washed in PBT and incubated in secondary antibody (goat, anti-rat, DyLight 549; Jackson) 1:500 in blocking buffer containing phalloidin (3 μl/100 μl) for 3 h at room temperature. After an additional washing step, samples were mounted on glass slides in Vectashield.
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