Several cell surface proteins were visualized by immunolabeling with primary rabbit antibodies against ATP5F1A or P2RY2 and secondary antibody conjugated to DyLight488 (NovusBio). The cells were fixed in cold methanol (− 20 °C, 5 min), blocked with BSA (5% in PBS, 1 h, room temperature-RT), and incubated with the primary antibody (1:100 dilution, 4 °C, overnight) in BSA (1% in PBS). The following day, the cells were incubated with the secondary antibody (1:2500 dilution, RT, 1 h, dark) in BSA (1% in PBS), and cured with ProLong Diamond antifade mountant with DAPI (RT, 24 h, dark) or DAPI solution (1 μg/mL, RT, 5 min, dark). Alternatively, the cells were fixed with paraformaldehyde (PFA) solution (2% in PBS, RT, 15 min) and permeabilized with Triton X-100 (0.5% in PBS, RT, 5 min). The blocking and incubation with antibody steps were the same as above. Images were acquired either with an inverted epi-fluorescence Eclipse TE2000-U microscope (Nikon Instruments Inc, Melville, NY) with a 20X air objective, or by confocal scanning with SoRa mode with a Nikon Eclipse Ti2 with a 40X water objective. The images were processed with Nikon software Denoise.ai and NIS-Elements AR Analysis 5.11.01.
Dylight 488
Manufactured by Novus Biologicals
Sourced in United States
DyLight 488 is a fluorescent dye that can be used to label proteins, peptides, and other biomolecules. It has an excitation maximum at 493 nm and an emission maximum at 518 nm, which falls within the green portion of the visible light spectrum. DyLight 488 is often used in applications such as immunofluorescence, flow cytometry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Denoise ai, by Nikon (1 mentions)
Eclipse ti2, by Nikon (1 mentions)
Normal horse serum, by Vector Laboratories (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Nestin, by Novus Biologicals (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Paraformaldehyde (pfa), by Biosesang (1 mentions)
A23204, by Thermo Fisher Scientific (1 mentions)
Sp8 lightning confocal microscope, by Leica (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Biorevo bz 9000 fluorescence microscope, by Keyence (1 mentions)
Blocking one p, by Nacalai Tesque (1 mentions)
Fluoview 1200 laser scanning confocal microscope, by Olympus (1 mentions)
6 protocols using dylight 488
1
Immunolabeling of Cell Surface Proteins
2
Immunofluorescence Characterization of Neural Cells
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Cells were fixed with ice-cold 4% paraformaldehyde (Biosesang, Gyeonggi, Korea) for 20 minutes at room temperature (RT) and permeabilized with 1% Triton X-100 (Sigma) for 5 minutes at RT. After washing with PBS, the cells were treated in a blocking solution containing PBS supplemented with 0.1% Triton X-100 (Sigma), 5% normal goat serum (Vector, Olean, NY, USA), and 5% normal horse serum (Vector) for 1 hour at RT. Then, the cells were incubated with primary antibodies overnight at 4°C; Nestin (1:2,500, Novus Biologicals, Littleton, CO, USA), STEM121™ (1:500, StemCells, Inc.), MAP-2 (1:200, Santa Cruz, Dallas, TX, USA), GFAP (1:2,000, Abcam). Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate (1:500, Thermo Fisher Scientific), Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate (1:500, Thermo Fisher Scientific), and Goat anti-Chicken IgG (H+L) Secondary Antibody [DyLight 488] (1:500, Novus Biologicals) were used as secondary antibodies for 2 hours at RT. The cells were treated with DAPI (1:1,000, Thermo Fisher Scientific) for 5 minutes at RT. After washing with PBS, the slides were mounted with Vectashield™ (Vector) and analyzed using an Axio observer Z1 as a fluorescence microscope (Zeiss, Jena, Germany).
3
Histological Analysis of Skin Samples
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For histological examination, mice and human skin explants after topical FNA treatment were fixed using 10% neutral-buffered formalin solution for 24 h. Skins were subsequently rinsed thrice with PBS, treated with 30% (w/v) sucrose for 16 h, and immersed into optimal cutting temperature (OCT) solution. Samples were then exposed to liquid nitrogen for cryosectioning. Finally, thin sample slices (15 μm) were stained with Hoechst33342, Annexin V AlexaFluor647 (Invitrogen™ A23204, 50-fold dilution) or anti-Keratin14 antibody (DyLight 488; Novus Biologicals NBP2-34675G, 5 μg/mL) according to the manufacturer’s protocol or with standard Hematoxylin & Eosin protocol.
4
Quantifying Microglial Activation in Arcuate Nucleus
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In another separate group of rats with or without siRNA injection (n=3–4/group), under deep anesthesia with isoflurane, rats were perfused through left ventricle with heparinized saline followed by 4% paraformaldehyde. Brain was removed, post fixed, and then placed in 30% sucrose. Brain sections with the ARCN (each section 15 µm thick) were cut with a cryostat according to a stereotaxic atlas and preserved in the cryoprotectant. The floating brain sections containing ARCN were blocked with 10% normal donkey serum and incubated with macrophage/microglia cell marker CD11b/c antibody conjugated with DyLight 488 (1:500, NB110‐40766, Novus Biologicals, Centennial, CO) overnight at 4°C. After washing, the sections were mounted on the glass slides and coverslipped with Vectashield mounting medium (Vector Laboratory, Burlingame, CA). The fluorescent images of CD11b/c within the ARCN were visualized and imaged using a Leica SP8 lightning confocal microscope (Leica, Germany).
5
C2C12 Myoblast Differentiation Assay
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C2C12 cells were seeded into a 12-well plate and cultured overnight. After cell adhesion, the culture medium was exchanged for differentiation medium, comprising DMEM supplemented with 2% heat-inactivated horse serum, with or without kynurenine, Trp, and Leu. The Leu-depleted medium, supplemented with 2% horse serum, contained 4.2 µM Leu, as the concentration of Leu in the horse serum was 181.4 µM. After incubation for seven days, the cell morphology was evaluated microscopically. The numbers of myotubes were counted in five fields per sample, and the average length was determined for 50 myotubes. Myotubes were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 min, followed by permeabilization with 0.2% Triton X-100 in PBS for 15 min. Cells were stained with anti-myosin heavy chain (MHC) antibody conjugated DyLight 488 (Novus biologicals; Centennial, CO, USA) for 24 h. The cells were counterstained with 4′6-diamidino-2-phenylindole dihydrochloride (DAPI) in PBS for 10 min. Fluorescent images were analyzed by using a BIOREVO BZ-9000 fluorescence microscope (Keyence, Osaka, Japan). The fusion index represented the number of nuclei in multinucleated MHC-positive cells, divided by the total number of nuclei in a field.
6
Cardiac Troponin T Immunostaining
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The cardiac cell sheet tissue was fixed with 4% paraformaldehyde, permeabilized with 0.2% Tween 20, and blocked with blocking agent (Blocking one-P, Nacalai Tesque). Immunostaining was performed using the following primary antibodies: mouse anti-cardiac troponin T (cTnT), cardiac isoform, mouse-mono (MS-295-P1, clone 13-11, LVC), and the following secondary antibodies: goat anti-Mouse IgG (H + L) secondary antibody; DyLight 488 (NBP1-75146, NOVUS), DAPI (D3571, Invitrogen). Confocal microscopy images were obtained using an Olympus FluoView 1200 laser scanning confocal microscope.
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