After Hepa1-6 cells growing on the coverslip were incubated with P. y. yoelii 265BY-RFP sporozoites for the indicated time, the cells were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilised with ice-cold methanol at −20 °C for 10 min, and blocked in 5% BSA and 0.3% TritonX-100/PBS for 1 h at room temperature. To detect autophagy, the cells were then stained with anti-LC3 (Abcam) and anti-LAMP1 (Abcam). They were then stained with Dylight 488 or Dylight 405-labeled secondary antibodies (Abcam), respectively. To investige the survival and replication of EEFs in the autolysosomes, the cells were stained with anti-LC3 (Abcam), anti-LAMP1 (Abcam) and anti-EdU (Abcam). To detect autolysosome maturation, the cells were stained with anti-LC3 (Abcam), anti-LAMP1 (Abcam) and Anti-Cathepsin D (Abcam), then, stained with Dylight 488, Dylight 405 or Dylight 649-labeled secondary antibody (Abcam), respectively. DNA was counterstained with DAPI in all of the above experiments. Coverslips were mounted with Dako Fluorescent Mounting Medium (Dako), and all images were acquired on a Leica DM 2000 confocal microscope and analysed using the Leica Application suite, version 2.3.0.
Dylight 488
Manufactured by Abcam
Sourced in United Kingdom, United States
DyLight 488 is a fluorescent dye used in various biological applications. It has an excitation maximum of 493 nm and an emission maximum of 518 nm, making it suitable for detection with common fluorescence imaging equipment. DyLight 488 can be used to label proteins, nucleic acids, and other biomolecules for analysis and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Ab96899, by Abcam (2 mentions)
P4170, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Facscalibur, by BD (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Facscanto 2, by BD (2 mentions)
Irdye800cw donkey anti mouse igg h l, by LI COR (2 mentions)
Goat anti mouse af647, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit af488, by Thermo Fisher Scientific (2 mentions)
Atto 565, by Abcam (2 mentions)
Lightning link rapid atto565, by Abcam (2 mentions)
Tub 1a2, by Merck Group (2 mentions)
Irdye680rd donkey anti rabbit igg h l, by LI COR (2 mentions)
Poly l lysine coated coverslip, by Merck Group (1 mentions)
Lsm 780 microscope, by Zeiss (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Ab32351, by Abcam (1 mentions)
P0099, by Beyotime (1 mentions)
Ab104139, by Abcam (1 mentions)
45 protocols using dylight 488
1
Analyzing Autophagy Pathway in Malaria Infection
2
Cell Visualization with Immunofluorescence Staining
3
Immunofluorescence Assay of Caspase-3 in HK-2 Cells
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We inoculated HK-2 cells (3 × 104 cells/mL) into a 6-well plate with cover glass until the cell confluence reached about 70%. After the cells received different treatments, we fixed the cells with a 4% paraformaldehyde fixation solution (P0099, Beyotime, China) and then rinsed them thoroughly with phosphate buffer (PBS, SH30256.01, Hyclone, USA) for 3 times. After permeating the cell membrane with 0.5% Triton X-100 (T109027, Aladdin, China) in each hole, we sealed the cells with 3% BSA blocking solution (B265993, Aladdin, China) for 30 min. They were then reacted with anti-Caspase-3 antibody (1:500, ab32351, abcam, UK) at 4 °C overnight. After the cells in each well were reacted with goat anti-rabbit IgG H&L (DyLight® 488, 1:500, ab96899, abcam, UK), they were reacted with DAPI (ab104139, abcam, UK). In the end, after mounting, the cover glass was placed under the inverted fluorescence microscope (Ts2-FC, Nikon, Japan) to observe the results.
4
Immunofluorescent Staining for Flow Cytometry
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The cells were dissociated and resuspended in 500 μl of 90% MeOH and left for 5–10 min at room temperature. The cells were then centrifuged, the liquid was removed and 500 μl of 0.1% Tween 20 was added to permeabilize the cells for 20 min at room temperature. The cells were centrifuged again, the liquid was removed, and 50 μl of Tween buffer and 0.5 μg of antibody was added to each tube and incubated in the dark for 30–45 min at room temperature. All antibodies were purchased from Ebioscience either directly conjagted to Alexa 488 or FITC; or if unconjugated, were directly conjugated to Dylight 488 (Abcam). The cells were then washed three times with FACs buffer then resuspended in FACs buffer. The cells were then measured using an LSRII flow cytometer (BD Biosciences). The results were analyzed using FlowJo software.
5
Immunofluorescence Imaging of Met Receptor in NP-Treated HepG2 Cells
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HepG2 cells were plated in a cell view cell culture dish (Greiner Bio-One North America, Inc., Monroe, NC, USA) at a density of 2.5 × 104 cells/compartment and incubated for 24 h. Next, the cells were exposed to NPs at a concentration of 50 μg ml–1 and incubated for an additional 24 h, after which the cells were washed twice to remove excess NPs and then fixed with 4% paraformaldehyde (PFA) for 10 min. Subsequently, the cells were blocked with 1% bovine serum albumin (BSA)/10% normal goat serum/0.3 M glycine in PBS for 1 h, followed by washing three times (5 min each). Immediately after washing, the cells were incubated with anti–Met hepatocyte growth factor receptor (HGFR) antibody (1/100 dilution, EP1454Y, Abcam, Cambridge, MA, USA) for 1 h at room temperature (RT) and then goat anti-rabbit IgG H&L (1/200 dilution, DyLight® 488, ab96883, Abcam) in the dark for 1 h at RT. Both incubations were followed by washing three times (5 min each) with PBS. Confocal microscopy was performed using a confocal laser-scanning microscope (LSM510 META, Carl Zeiss Inc., Jena, Germany). All images were acquired using a 63 × 1.4 Plan-Apochromat oil immersion objective (Carl Zeiss).
6
Phenotypic Characterization of Canine BMSCs
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Phenotypic characterization of canine BMSCs was performed as described.18 Adherent cells were dissociated with 0.05% trypsin‐ethylenediaminetetraacetic acid (Life Technologies) and resuspended in DMEM containing 10% fetal bovine serum. The cells were washed once with phosphate‐buffered saline (Life Technologies), and after centrifugation the phosphate‐buffered saline was removed and replaced with phosphate‐buffered saline containing 2% canine serum (AbD Serotec, Oxford, UK). The cells were placed on ice for 20 minutes, followed by incubation for a further 20 minutes on ice with monoclonal antibodies against cluster of differentiation 11b (CD11b; AbD Serotec), CD29‐PE (Abcam, Cambridge, UK), CD44‐APC (Biolegend, San Diego, CA), CD45‐eFlour (ebioscience, San Diego, CA), and CD90‐APC (ebioscience). The CD11b antibody was detected using goat polyclonal secondary antibody to mouse immunoglobulin G heavy and light chains (DyLight 488; Abcam). Isotype‐identical antibodies were used as controls. Flow‐cytometric analyses were performed using the Gallios system (Beckman Coulter, Danvers, MA). Propidium iodide (Sigma‐Aldrich, St. Louis, MO) was used to exclude dead cells from analyses. Each sample was assessed at least in triplicate. Data were analyzed using Kaluza software (Beckman Coulter).
7
Immunoglobulin Profiling of Strep A-Reactive Plasma Cells
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To clarify the immunoglobulin class of specific antibodies to Strep A produced by plasma cells in the tonsils, double immunofluorescence staining was performed using biotinylated Strep A and DyLight 488‐labeled goat antibodies against human α‐chain, γ‐chain or μ‐chain (Abcam, Cambridge, MA, USA). After a brief dip in running water, prefixed frozen sections of the tonsils were treated with TBS containing 5 μg/mL proteinase K at room temperature for 15 min. The sections were incubated with biotinylated Strep A (50 μg/mL) overnight at room temperature, followed by incubation with a mixture of Alexa Fluor 488 (red)‐labeled streptavidin (diluted 1:300; Molecular Probes) and DyLight 488 (green)‐labeled goat antibodies against human α‐chain (diluted 1:900); γ‐chain (diluted 1:300), or μ‐chain (diluted 1:300) at room temperature for 1 hr. The nuclei were stained blue with DAPI.
By examining consecutive sections, (i) the ratio of immunoglobulin classes to total plasma cells (the sum of IgA + IgG + IgM plasma cells) in the tonsils and (ii) the percentage of Strep A‐reactive plasma cells of the respective immunoglobulin class were evaluated.
By examining consecutive sections, (i) the ratio of immunoglobulin classes to total plasma cells (the sum of IgA + IgG + IgM plasma cells) in the tonsils and (ii) the percentage of Strep A‐reactive plasma cells of the respective immunoglobulin class were evaluated.
8
Visualization of RNA Synthesis in DAOY Cells
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DAOY cells were seeded in chamber slides (0,3 cm2/well; 5×103 cells/well) and at the indicated time intervals p.i. processed as previously described [47 (link)]. Rabbit anti-POLR1A (Abcam; 1:200) and chicken anti-NS3 (a kind gift from Dr. M. Bloom, NIAID, NIH; 1:5000) antibodies were used. As the secondary antibodies, anti-rabbit DyLight 594 (Abcam; 1:500) and anti-chicken DyLight 488 (Abcam; 1:500), were used. In the case of metabolic labelling of nascent RNA, the Click reaction was performed in situ before the blocking step. 10 μM Picolyl biotin azide was used for the detection of incorporated 5-EU. For subsequent fluorescent labelling, streptavidin conjugated with DyLight 549 was used (VectorLabs; 1:500). Slides were eventually mounted in Vectashield mounting medium (VectorLabs). The Olympus Fluoview FV10i confocal microscope was used for imaging and subsequent export of images was done in FV10-ASW software (v.1.7).
9
Immunophenotyping of Tumor Cells by FNAB
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A perCP/Cy5.5 conjugated EpCAM mouse anti-human monoclonal antibody (VU-1D9, abcam) and a FITC conjugated CD44 mouse anti-human (clone G44-26, BD pharmingenTM) and their respective isotype controls, mouse IgG1 monoclonal MOPC-21 (perCP/Cy5.5, abcam) and Dnk pAb to Ms IgG (Dylight®488, abcam) were diluted with RPMI media to a working solution 1:200. FNAB tumor samples were loaded into nine separate channels. The antibody solution containing both the EpCAM and CD44 was perfused into three channels. The isotype control antibodies, used to evaluate for nonspecific binding, were perfused into three other channels. The last three channels contained media only to serve as background auto-fluorescence controls for tissue. One channel was not needed and therefore not used for this experiment. A flow rate of 125 μL/hr was used for all channels. Fluorescent imaging was performed by a Zeiss Axio ObserverZ.1/ApoTome.2 inverted scope using a 378HE Green Fluorescent Protein (GFP) filter (excitation: 450-490nm /emission: 500-550nm) and a 45 Texas Red (TR) filter (emission: 540-580/excitation: 593-668nm) over a 24-hour period.
10
STAT3 Regulation in Glioma Stem Cells
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GSCs were infected with lentivirus carrying Flag-STAT3 or Flag-STAT3-4KR mutations and then infected or not with lentivirus carrying pLKO.1-shOTULIN. GSCs were plated on cover glasses coated with Matrigel (Corning, 354277) in a 24-well plate at a density of 1 × 105 cells/well and incubated overnight. Cells were fixed in 4% paraformaldehyde for 20 min, and then blocked in 10% FBS with 0.3% Triton X-100 in PBS for 20 min at room temperature, followed by incubation with primary antibody Phospho-Stat3(Tyr705) (1:100, 9145S, CST) overnight at 4°C. Then cells were incubated with the secondary antibody labeled with DyLight 488 (1:100, ab96899, Abcam) for 1 h at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired on a Zeiss LSM880 system; the acquisition software was Zen 2.1 SP2.
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