Immunofluorescence assay was performed according to the protocol described previously (43 (link)). Briefly, the attached cells were harvested at indicated stages, fixed with 4% paraformaldehyde, permeabilized in 0.4% Triton X-100 (Sigma-Aldrich) for intracellular antigens, blocked in 10% normal goat serum (Vector Laboratories), and then stained with antibodies to FAM46B (Cat No. 23149-1-AP, Proteintech, 1:200). Antibody labeling was visualized using DyLight 488-conjugated secondary antibodies (all from Jackson ImmunoResearch Laboratories, 1:1000). Nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich, 1:2000). A Zeiss Observer microscope was used for slide observation and image capture.
Dylight 488 conjugated secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
DyLight 488-conjugated secondary antibodies are fluorescent-labeled antibodies that bind to the Fc region of primary antibodies. DyLight 488 is a fluorescent dye that emits light in the green region of the visible spectrum when excited. These antibodies are commonly used in immunoassays and microscopy techniques for the detection and visualization of target proteins or molecules.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (2 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Fam46b, by Proteintech (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
Anti ttyh1, by Cusabio (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Epifluorescence microscopy, by Leica (1 mentions)
Fluoview fv1000 confocal microscope, by Olympus (1 mentions)
Ab5076, by Abcam (1 mentions)
Ab5622, by Abcam (1 mentions)
Mkb 2213, by Vector Laboratories (1 mentions)
Af2535, by R&D Systems (1 mentions)
Anti lyve 1 antibody, by Abcam (1 mentions)
Sp2 confocal microscope, by Leica (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Tcs sp5 confocal laser scanning microscope, by Leica (1 mentions)
Lab tek 2 chamber slide, by Thermo Fisher Scientific (1 mentions)
9 protocols using dylight 488 conjugated secondary antibody
1
Immunofluorescence Assay for FAM46B Localization
2
Immunofluorescence Assay for TTYH1 Expression
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U2OS cells transfected with siRNAs grown on coverslips were fixed with 4% paraformaldehyde for 20 min at room temperature and were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS). The cells were blocked with 3% bovine serum albumin and then incubated overnight at 4 °C with anti-TTYH1 (1:200; Cusabio technology LLC, Houston, TX, USA) antibody. After rinsing in cold PBS, the cells were treated with DyLight 488-conjugated secondary antibodies (1:500; Jackson Labs, Harbor, ME, USA) for 1 h at room temperature. The cells were washed, mounted, and observed under an A1 confocal microscope (Nikon, Minato-ku, Tokyo, Japan).
3
Visualizing FGF2-induced RSK2 and FGFR2 localization
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Cells seeded onto glass coverslips were serum-starved overnight and stimulated with FGF2 for 20 min. Specimens were fixed with 2 % paraformaldehyde in PBS and permeabilized with 0.1 % Triton X-100 in PBS for 1 min. After blocking with 3 % BSA in PBS, cells were incubated with specific primary antibodies: anti-RSK2 (1:75) and anti-FGFR2 (1:100) for 1 h. Cells were then stained with AffiniPure DyLight 549-conjugated or DyLight 488-conjugated secondary antibodies (Jackson ImmunoResearch). Distribution of analysed proteins was examined by using a fluorescence microscope ZEISS AxioVert 200.
4
Immunostaining of Olig2-Expressing Cells
5
Immunofluorescence Staining of Mouse Brain Tissue
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Mice were sacrificed 7 and 35 days post‐dMCAO. Coronal slices (25‐μm‐thick) were prepared for immunofluorescence staining as previously described.18 The following primary antibodies were used: rabbit anti‐NeuN (EMD Millipore), rabbit anti‐microtubule‐associated protein 2 (MAP2; sc‐20,172; Santa Cruz), mouse anti‐200kD neurofilament heavy (NF200; MAB5262; Millipore Sigma), rabbit anti‐beta‐amyloid precursor protein (β‐APP; 512700; Fisher Scientific), rabbit anti‐myelin basic protein (MBP; ab5622; Abcam), goat anti‐Iba1 (ab5076; Abcam), goat anti‐CD206 (AF2535; R&D Systems), rat anti‐CD16 (553142; BD Pharmingen), and mouse anti‐SMI‐32 (ab50761; Abcam). Samples were blocked in 5% normal donkey serum (Jackson ImmunoResearch) to reduce nonspecific binding. When a mouse primary antibody was used, a mouse‐on‐mouse (M.O.M) blocking reagent (MKB‐2213; Vector Laboratories) was used to avoid cross‐reactivity. Donkey Cy3‐ or DyLight 488‐conjugated secondary antibodies (Jackson ImmunoResearch) were used. Images were obtained using an Olympus FluoView FV1000 confocal microscope (Olympus America) and analyzed with ImageJ. Cell numbers were calculated from two random microscopic fields in each mouse brain.
6
Visualizing Blood and Lymph Vessels
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Endothelial blood vessels were stained with a hamster monoclonal anti-CD31 antibody (Chemicon International, Temecula, USA; MAB1398Z) and endothelial lymph vessels with a rabbit polyclonal anti-LYVE-1 antibody (Abcam, Cambridge, UK; ab14917). Nuclei were Hoechst 33342-labeled (Sigma Aldrich, Taufkirchen, Germany). DyLight488-conjugated secondary antibodies (donkey) were obtained from Jackson ImmunoResearch (Pennsylvania, USA). Primary and secondary antibodies were diluted 1∶100 in PBS/0.3% Triton-X-100.
7
Visualizing Membrane and Intracellular α-Sarcoglycan
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To visualize membrane-resident α-sarcoglycan, immunofluorescence experiments were performed on intact cells. V247M cells, seeded and grown on poly-lysine-treated glass coverslips, or human myoblasts seeded, grown and differentiated on gelatin/fibronectin-treated glass coverslips, at the end of specific drug treatments, were chilled at 4°C for 30 min, washed with cold PBS supplemented with Ca2+ and Mg2+. Cells were subsequently incubated at 4°C with either the mouse monoclonal or the rabbit polyclonal antibody specific for α-sarcoglycan, recognizing an extracellular epitope. After washing, cells were incubated with the specific DyLight 488-conjugated secondary antibody (Jackson). At the end of incubation, cells were washed and fixed with 4% PFA.
To visualize intracellular α-sarcoglycan, immunofluorescence experiments were performed on permeabilized cells. V247M cells cultivated as above described, were first fixed with 4% PFA and subsequently permeabilized with 0.5% Triton X-100 for 15 min. Incubation with primary and secondary antibodies was performed as described above. Cells were then examined with a Leica SP2 confocal microscope.
To visualize intracellular α-sarcoglycan, immunofluorescence experiments were performed on permeabilized cells. V247M cells cultivated as above described, were first fixed with 4% PFA and subsequently permeabilized with 0.5% Triton X-100 for 15 min. Incubation with primary and secondary antibodies was performed as described above. Cells were then examined with a Leica SP2 confocal microscope.
8
Visualizing ZEB1 Expression in A549 Cells
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A549 cells were seeded on glass coverslips in an environment containing 5% CO2 in air at 37 °C and then incubated in 10 nM neutrophil elastase and 20 μM EGCG for an additional 24 h. The coverslips were gently washed in PBS, fixed in 3.7% paraformaldehyde for 15 min, permeabilized in PBS containing 0.1% Triton X-100 for 20 min and blocked in PBS containing 3% BSA for 30 min. The fixed cells were incubated in an anti-human zinc finger E-box-binding protein-1 (ZEB-1) antibody (Cell Signaling Technology, Beverly, MA, USA) overnight at 4 °C in a humidified chamber. Then, the cells were washed and incubated in the corresponding DyLight 488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at 37 °C. After washing in PBS, the cells were incubated in Hoechst 33342 (1 μg/ml, Sigma-Aldrich) for 5 min at room temperature. After several washes, the immunostained specimens were observed under a TCS-SP5 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany).
9
Mesangial Cell Fibronectin Induction
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Mesangial SV40-Mes13 cells (2 × 104 cells/well) were seeded onto 8-well Lab-Tek II chamber slides (NUNC, Rochester, NY). After serum starvation, cells were treated with OA/BSA (200 μM) alone or in combination with 200 μg/mL of OF or CM. After 24 h incubation, mesangial cells were washed with cold PBS, fixed with acetone/methanol (1/1; v/v), and then stained. After 1 h blocking with 3% BSA in PBS with 0.1% Triton X-100 (PBST), the cells were incubated with a primary antibody (rabbit anti-mouse fibronectin antibody, Epitomics, CA, Burlingame, USA) overnight, washed with PBS, and incubated with the DyLight 488-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 2 h. The slides were mounted with 87% glycerol and imaged using a DeltaVision® Core live-cell microscope (Applied Precision Inc., WA, USA).
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