Dissected larval brains with attached CPS and intact pharyngeal nerves were fixed for 1 hr in paraformaldehyde (4%) in PBS, rinsed with PBS-T and blocked in PBS-T containing 5% normal goat serum. For antibody stainings of Gr-GAL4 >10xUAS-mCD8::GFP primary antibody were conjugated goat anti-GFP (1:500, Abcam, ab6662), mouse anti-fasciclin2 (1:500, DSHB) and mouse anti-22C10 (1:500, DSHB) and the secondary antibody was anti-mouse Alexa Flour 568 (1:500, Invitrogen). Brains were rinsed with PBS-T and mounted in Mowiol (Roth, 0713). For antibody stainings of Gr43a-GAL4 > 10xUAS-mCD8::GFP primary antibody were rabbit anti-GFP (1:500, Abcam, ab6556), mouse anti-fasciclin2 (1:500, DSHB) and mouse anti-22C10 (1:500, DSHB) and the secondary antibody were anti-rabbit Alexa Flour 488 (1:500, Invitrogen) and anti-mouse Alexa Flour 568 (1:500, Invitrogen). Brains were rinsed with PBS-T and dehydrated through an ethanol-xylene series and mounted in DPX. Imaging was carried out using a Zeiss LSM 780 confocal microscope with a 25x objective (Zeiss).
Alexa flour 568
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 568 is a fluorescent dye produced by Thermo Fisher Scientific. It is a synthetic dye with a peak excitation wavelength of 578 nm and a peak emission wavelength of 603 nm, falling within the red-orange region of the visible light spectrum. Alexa Fluor 568 is commonly used in various biological and biomedical applications, including fluorescence microscopy, flow cytometry, and immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Alexa flour 488, by Thermo Fisher Scientific (16 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Vectashield mounting medium, by Vector Laboratories (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Western blot detection reagent ecl solution, by Advansta (2 mentions)
Alexa flour 647, by Thermo Fisher Scientific (2 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (2 mentions)
Anti ampa receptor 1, by Cell Signaling Technology (2 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (2 mentions)
Chicken anti gfp, by Abcam (2 mentions)
Lsm 780, by Zeiss (2 mentions)
H2ax ser 139, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Triton x, by Merck Group (1 mentions)
Cyclin a h 432, by Santa Cruz Biotechnology (1 mentions)
Geltrex coated cover slips, by Thermo Fisher Scientific (1 mentions)
Scanr screening station, by Olympus (1 mentions)
35 protocols using alexa flour 568
1
Larval Brain Dissection and Immunostaining
2
Immunofluorescence Staining for DNA Damage and Cell Cycle Analysis
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For immunofluorescence staining, cells grown on geltrex-coated coverslips (Invitrogen, Carlsbad, CA, USA, cat.no. 14113-202) and fixed with 4% paraformaldehyde (PFA) (Sigma-Aldrich, St. Louis, MI, USA, cat.no. P6148), permeabilized using 0.25% Triton-X (Sigma-Aldrich, St. Louis, MI, USA, cat.no. T9284) followed by blocking for 30 min in 10% Bovine Serum Albumin (BSA) (Bovine Albumin Fraction V, 7.5% solution; Thermo Fisher Scientific, Waltham, MA, USA, cat.no. 15260037). Cells were incubated with respective primary antibodies (H2AX (Ser139) (Merck Millipore, Burlington, MA, USA, cat.no. 05-636); Cyclin A (H-432) (Santa Cruz Biotechnologies, Dallas, TX, USA, cat.no. sc-751) in 2% BSA (Bovine Albumin Fraction V, 7.5% solution; Thermo Fisher Scientific, Waltham, Massachusetts, cat.no. 15260037) for 2 h followed by incubation with appropriate secondary conjugated antibodies (Alexa Flour 488 (Invitrogen, Carlsbad, CA, USA, cat.no. A11029); Alexa Flour 568 (Invitrogen, Carlsbad, CA, USA, cat.no. A11036). Nuclei were counterstained with 4,6-Diamidino-2-Phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MI, USA, cat.no. D9542-10MG). Automated imaging and analysis were performed using Olympus Scan-R screening station equipped with Scan-R analysis software as described previously [29 (link)].
3
Immunofluorescence assay for JEV detection
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N2a cells were grown on the coverslip in a 24-well plate. After 24 h, cells were first infected with JEV (MOI = 5), and after 3 hpi, cells were transfected with amiRNAs. The culture medium was removed at 48 hpi. The cells were washed thrice with PBS and fixed with 4% paraformaldehyde at RT for 15 min. Cells were permeabilized in 0.25% Triton X-100 for 10 min at RT and blocked with PBS containing 2% bovine albumin sera plus 0.4% Triton X-100 at RT for 30 min. An in-house rabbit polyclonal to JEV nonstructural 1 (NS1) antibody at a dilution of 1:1,000 was added and incubated at room temperature for 1 h. Cells were washed thrice with PBS, as above, and then incubated with secondary antibody (goat anti-rabbit immunoglobin G conjugated with Alexa flour 568; Invitrogen) diluted 1:500 with PBS for 1 h at RT in the dark. After washing multiple times with PBS, cells were then washed thrice with PBS and mounted with a slow-fade gold antifade reagent with 4′, 6-diamidino-2-phenylindole (Invitrogen).
4
RSV-A2-GFP Infection and Phenotyping
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100,000 cells were seeded onto a Transwell insert infected 3 h later with RSV-A2-GFP at an MOI of 0.1. Uninfected control cells were incubated with the respective volume of PBS. After 20 h cells where harvested and fixed by 2% paraformaldehyde for 10 min at RT, centrifuged for 5 min at 1,500 rcf and washed once in PBS. Fixed cells were permabilized by PBS+0.5% Triton X-100 for 15 min at RT, followed by washing once in PBS. Fixed and permabilized cells were stained for p63 (Abcam ab124762) and ITGA6 (Millipore MAB1378) at a dilution of 1∶200 for 45 min, washed once, and incubated with an Alexaflour 568 and Alexaflour 647 conjugated secondary antibody (Invitrogen) at 1∶1000 for 30 min. Flow cytometry was performed in PBS using a Fortessa cytometer.
5
Neural Stem Cell Characterization Protocol
6
Whole-Mount Immunofluorescence of Embryos
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Embryos were fixed in 4% paraformaldehyde in PBS for 1 h at room temperature, washed with PBS, fixed again with 100% methanol overnight at 4°C, and then washed with 75% methanol plus 25% PBST, 50% methanol plus 50% PBST, 25% methanol plus 75% PBST, and 100% PBST. Fixed embryos were permeabilized by incubation with 0.1 mg/ml proteinase K in PBSX (0.1% Triton X-100 in PBS) for 15 min at room temperature and then washed with PBSDX (1% DMSO and 0.3% Triton X-100 in PBS) three times. Permeabilized embryos were blocked in blocking solution (2% fetal bovine serum and 2 mg/ml BSA in PBSX) for 15 min at room temperature, reacted with mouse anti–type II collagen antibody (Thermo Fisher Scientific) and rabbit anti-calnexin antibody (ADI-SPA-865; Enzo Life Sciences) in blocking solution for two nights at 4°C, washed five times with PBSDX, reacted with goat anti–mouse IgG heavy and light chains (Alexa Flour 568) and goat anti-rabbit IgG heavy and light chains (Alexa Flour 488; Invitrogen) for 1 h at room temperature, and then washed with PBST.
7
Novel Compound Synthesis and Assays
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1-Methyl-4-phenylpyridium (MPP⁺) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dichlorofluorescein diacetate (DCF-DA), DAPI (4′,6-diamidino-2-phenylindole), Alexa Floure 488, and Alexa Flour 568 were obtained from Invitrogen (Portland, OR, USA). Western blot detection reagent (ECL solution) was supplied by Advansta Inc. (San Jose, CA, USA). MHY2699 was synthesized by Prof. Hyung Ryong Moon (College of Pharmacy, Pusan National University, Busan, Korea), as described elsewhere [18 (link)].
8
Quantifying Protein Aggregates in Drosophila
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Flies were fixed for 20 min with 3.7% formaldehyde in PBS, and after fixation, hemi-thoraces were dissected. Samples were rinsed three times with 0.2% Triton X-100 in PBS (PBST) and blocked for 1 h at room temperature in 3% BSA in PBST. Primary antibodies (anti-FK2, mono- and polyubiquitinylated conjugates monoclonal antibody, BML-PW8810, Enzo) were diluted 1:250 in 5% BSA in PBST and samples were incubated overnight at 4 °C. Samples were rinsed thrice with PBST and incubated in a mix of 1:250 diluted secondary antibodies (anti-mouse AlexaFluor-488, Invitrogen) and a stain (phalloidin AlexaFlour-568, Invitrogen) in 5% BSA in PBST for 3 hours at room temperature. Samples were rinsed three times with PBST and mounted in Vectashield mounting medium (Vector Labs) and imaged using a Zeiss single point LSM 5 exciter confocal microscope. Protein aggregate and total muscle areas were quantified using ImageJ using the analyze particle features and the aggregate values were normalized by the area of muscle.
9
Quantifying Primary Cilia in MSCs
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The cells are cultured in in MesenPRO medium, and the MSCs cells were fixed after 6 days, when they had become confluent. Primary cilia were stained with anti-acetylated α-tubulin (from Abcam ab24610) and rabbit anti-γ -tubulin antibody (from Abcam ab16504). Mitotic cells were stained to detect phospho-histone H3 (stain from Abcam ab5176) and subsequently with the appropriate fluorochrome-conjugated secondary antibodies (Alexa Flour 488 and Alexa Flour 568, Invitrogen). Nuclei were stained with DAPI (Invitrogen, 1:600) and counted. Slides were mounted with VECTASHIELD reagent (Vector Labs). Images were captured using a Cool Snap ES camera (Photometrics) with MetaVue software (Molecular Devices) or a Nikon digital camera and analyzed and Image J software.
10
Immunolabeling Mitochondria and Aggregates in Drosophila
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