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 488
Manufactured by Jackson ImmunoResearch
Sourced in United States, Panama, United Kingdom, France
DyLight 488 is a fluorescent dye used in immunology and molecular biology applications. It has an excitation maximum at 493 nm and an emission maximum at 518 nm, allowing detection in the green fluorescence channel. The dye can be conjugated to various biomolecules, such as antibodies, proteins, and nucleic acids, for labeling and detection purposes.
Automatically generated - may contain errors
Lab products found in correlation
Dylight 549, by Jackson ImmunoResearch (18 mentions)
Dylight 594, by Jackson ImmunoResearch (15 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (15 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (9 mentions)
Dylight 649, by Jackson ImmunoResearch (8 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (7 mentions)
Lsm 710 confocal microscope, by Zeiss (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
Mouse monoclonal anti human glucagon, by Merck Group (5 mentions)
Polyclonal goat anti somatostatin, by Santa Cruz Biotechnology (5 mentions)
649 conjugated secondary antibodies, by Jackson ImmunoResearch (5 mentions)
Polyclonal guinea pig anti porcine insulin, by Agilent Technologies (5 mentions)
Triton x 100, by Merck Group (5 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (5 mentions)
Alexa 488, by Thermo Fisher Scientific (4 mentions)
Alexa 647, by Jackson ImmunoResearch (4 mentions)
Alexa 555, by Thermo Fisher Scientific (4 mentions)
Hoechst 33342, by Merck Group (3 mentions)
Vectashield h 1000, by Vector Laboratories (3 mentions)
Tcs sp5, by Leica (3 mentions)
111 protocols using dylight 488
1
Immunohistochemical Staining of Mouse Brain
2
Visualizing Lymphatic Vessels in Tissues
3
Whole Mount Lymphatic Vessel Labeling
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To visualize lymphatic vessels within their microenvironment and study specific features that were identified during live imaging, pinnas were fixed in 1 % paraformaldehyde in PBS pH 7.4 and labeled as previously described using whole mount technique [13 (link)]. Whole mount mouse cornea and pinna was stained with antibodies to LYVE-1 (11-034, AngioBio, Del Mar, CA), DAPI (Sigma–Aldrich) and the appropriate secondary antibody: using the fluorochromes Alexa488, (ThermoFisher Scientific) and DyLight488 (Jackson ImmunoResearch Laboratories, West Grove, PA). Fixed and labeled whole mounts were mounted in Vectashield H-1000 (Vector Laboratories, Burlingame, CA).
4
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.
5
Immunohistochemistry of CD138 and CXCR4
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For immunohistochemistry, the following antibodies were used: anti-CD138 mouse (B-A38) monoclonal antibody (Cell Marque, CA, USA) and anti-CXCR4 rabbit polyclonal antibody (Abcam). After deparaffinization and rehydration, the slides were placed in a pressure cooker in 0.01 M citrate buffer (pH 6.0) and were heated for 7 min. Incubation with the different antibodies was carried out overnight at 4°C. Detection was performed with DAKO en vision system according to the manufacturer′s protocol. For double immunofluorescence, primary antibodies were detected by incubation with the following secondary antibodies: donkey anti-rabbit conjugated with Dylight 488 (Jackson ImmunoResearch, Suffolk, UK) and donkey anti-mouse conjugated with Cy5 (Jackson ImmunoResearch). After incubation of slides with conjugated secondary antibody for 30 min, slides were counterstained and mounted with mounting medium (Vectashield, Vector laboratories, Burlingame, CA, USA).
6
Immunohistochemistry of Cardiac Tissue
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Hearts were fixed overnight in 4% paraformaldehyde at 4°C, washed with PBS several times, equilibrated in 30% sucrose in PBS and frozen in OCT (Tissue-Tek) for cryosectioning. Immunohistochemistry was performed on 10 µm cryosections by using MF20 (1 : 1, DSHB), anti-BrdU (1 : 50, Accurate) and anti-GFP (1 : 200, GFP-1020; Aves Labs) primary antibodies; and DyLight 488 (1 : 50, Jackson Immuno Research Laboratories), Cy5 (1 : 200, Jackson Immuno Research Laboratories) and Cy3 (1 : 200, Jackson Immuno Research Laboratories) as secondary antibodies.
7
Immunofluorescence and western blot
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Highly pre-adsorbed αmouse, αrabbit DyLight 488 and αguinea pig 649 were obtained from Jackson ImmunoResearch (West Grove, PA) and used at 1:500. Anti-mouse and αrabbit Atto 425 conjugated secondary antibody was obtained from Rockland, Inc. (Gilbertsville, PA) and used at 1:250. Horseradish peroxidase conjugated secondary antibodies (αmouse and αrabbit) were obtained from Jackson ImmunoResearch and used at 1:20,000.
8
Immunohistochemistry of Coronal Brain Sections
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Coronal brain sections (25 μm thick) were used for immunohistochemistry. Sections were washed three times in phosphate‐buffered saline (PBS) solution, blocked with 10% goat serum for 1 hour, followed by 1‐hour incubation in a primary antibody (listed in supplementary file, Table S2 ) at 37°C and overnight at 4°C. The following day, brain sections were washed three times in PBS containing 0.3% Triton‐X 100 (PBST) and then incubated with a species‐specific secondary antibody conjugated with DyLight 488 or DyLight 594 (Jackson ImmunoResearch Laboratories, West Grove, PA). Sections were then mounted with Fluoromount‐G (Southern Biotech, Birmingham, AL). Images were captured using confocal microscopy (Olympus America, Center Valley, PA).
9
Immunohistochemistry with Fluorescent Labeling
10
GnRH3 expression profiling by in situ hybridization
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Whole-mount in situ hybridization was performed using digoxigenin-labeled antisense RNA probes followed by detection with anti-digoxigenin alkaline phosphatase-conjugated antibody as described previously [27] (link). The pGEM-T-gnrh3 plasmid was linearized with NcoI before being used as a template for in vitro transcription with SP6 RNA polymerase. After in situ hybridization, staining and mounting, images were captured with a digital camera (Coolpix 990, Nikon).
For double staining, after in situ hybridization, digoxigenin-labeled gnrh3 antisense RNA probe was first reacted with mouse anti-Digoxin conjugated DyLight 488 (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) followed by immunochemical reaction with rabbit anti-GFP antibody (sc-8334, Santa Cruz, CA, USA) and detection with Alexa Fluor 647-conjugated to donkey anti-rabbit IgG antibody (Invitrogen Corporation, Carlsbad, CA, USA). The signals were observed using a Leica TCS-SP5-MP-SMD confocal system (Leica Microsystems Wetzlar, Wetzlar, Germany).
For double staining, after in situ hybridization, digoxigenin-labeled gnrh3 antisense RNA probe was first reacted with mouse anti-Digoxin conjugated DyLight 488 (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) followed by immunochemical reaction with rabbit anti-GFP antibody (sc-8334, Santa Cruz, CA, USA) and detection with Alexa Fluor 647-conjugated to donkey anti-rabbit IgG antibody (Invitrogen Corporation, Carlsbad, CA, USA). The signals were observed using a Leica TCS-SP5-MP-SMD confocal system (Leica Microsystems Wetzlar, Wetzlar, Germany).
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