Cells were fixed with 3.7% formaldehyde in PBS for 10 min at room temperature and permeabilized with 0.2% Triton™ X-100 in PBS for 10 min. After blocking with 2% bovine serum albumin in PBS (PBA) for 30 min, the samples were incubated with the appropriate primary antibody (1:200) in 2% PBA for 1 h at room temperature. Bound antibody was visualized with Alexa Fluor® 488- or Alexa Fluor® 568-conjugated antibody (1:200, Life Technologies, Carlsbad, CA). DAPI (Sigma-Aldrich) was used as a nuclear counterstain.
Alexa fluor 568 conjugated antibody
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, Germany, United States
Alexa Fluor 568-conjugated antibody is a fluorescent labeling reagent. It is used to fluorescently label proteins or other biomolecules for detection and visualization in various applications, such as immunoassays, flow cytometry, and fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade mountant, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Hoechst 33258, by Thermo Fisher Scientific (2 mentions)
Anti rps6, by Proteintech (2 mentions)
Antihemagglutinin anti ha, by Merck Group (2 mentions)
Tcs sp8 sted, by Leica (2 mentions)
Alexa fluor 488 conjugated antibody, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Orca er camera, by Hamamatsu Photonics (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (1 mentions)
Anti human mpo antibody, by Agilent Technologies (1 mentions)
Crossbeam 540, by Zeiss (1 mentions)
Prolong gold antifade with dapi, by Thermo Fisher Scientific (1 mentions)
Tcs sp5, by Leica (1 mentions)
Goat serum, by Agilent Technologies (1 mentions)
Ultraview vox spinning disk, by PerkinElmer (1 mentions)
Eclipse ti e, by Nikon (1 mentions)
Stellaris 5 confocal microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
14 protocols using alexa fluor 568 conjugated antibody
1
Immunofluorescence Staining Protocol
2
Visualizing HBV Envelope Protein Localization in Hepatocytes
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Huh-7 and HepG2 cells were seeded in chamber slides (Lab TekTM distributed by Fisher Scientific GmbH, Schwerte, Germany), transfected with pSVL and pSVM plasmids or treated with 10 nM thapsigargin for 72 hours. Immunofluorescence was performed as previously described [52 (link)]. Primary antibody against BiP was purchased from Abcam (Cambridge, UK) and secondary AlexaFluor 568-conjugated antibody from Life Technologies. The HBV envelope proteins were detected with the following antibodies: the mouse monoclonal antibody MA 18/07 (anti-preS1), used to detect the LHBs protein [53 (link)], and HB01 (anti-SHBs) used to detect MHBs protein, were a kind gift of Dr. Glebe and Dr. Aurelija Zvirbliene, (Institute of Biotechnology, University of Vilnius, Lithuania), respectively. To examine the ER compartment, Huh-7 and HepG2 cells were stained with 1 μM ER-tracker Red (Glibenclamide BODIPY TR, Life Technologies) according to the manufacturer's instructions and detected by Immunofluorescence. The fluorescence was visualized with a Nikon microscope at 630 × magnification and acquired with a Hamamatsu ORCA-ER camera (model C4742–80) under the same setting. Obtained data were analysed with ImageJ software v 1.43u.
3
p120-Catenin and F-actin Co-immunostaining
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p120-Catenin/F-actin co-immunostaining was performed as already described48 (link). Cells were rinsed, fixed, permeabilized and primary rabbit monoclonal anti-p120 catenin antibody (Abcam, ab92514) (1/100 final) was added and incubated for 1 h at room temperature. Corresponding secondary antibody (Alexa Fluor 568-conjugated antibody, ThermoFisher Scientific, ref A11036) (1/250 final) was added for 1 h at room temperature. After rinsing, 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (ThermoFisher Scientific, D1306) staining was performed for 10 min at room temperature at 0.2 µg/mL (final concentration). After rinsing, slides were mounted in Vectashield mounting medium without DAPI (Eurobio/Abcys).
4
Visualizing Neutrophil Extracellular Traps
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After neutrophil incubation with the respective biofilm, NET formation regarding their structure associated with the biofilm was visualized by immunocytochemistry (ICC) or scanning electron microscope (SEM). For ICC, the cells were fixed with 4% paraformaldehyde (PFA). NET visualization was performed as previously described [53 ]. In short, after permeabilization and blocking, NETs were primary stained with a rabbit polyclonal anti-human MPO antibody (1:300 in blocking buffer, Dako A0398). The secondary staining was performed using a goat anti-rabbit Alexa Fluor 568-conjugated antibody (1:500 in blocking buffer, Thermo Scientific A11031). The samples were embedded in ProlongGold® antifade with DAPI (P36931, Molecular Probes) and analyzed by CLSM (TCS SP5, Leica). For SEM, the cells were fixed with 0.1% glutaraldehyde and 4% PFA diluted in 200 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). The samples were dried and sputtered as previously described [54 ] and analyzed by SEM (Crossbeam 540, Zeiss). Both techniques allowed the qualitative observation of NETs.
5
Laser-induced Lysosomal Dynamics in MCF7 Cells
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MCF7 eGFP-LC3 cells grown in MatTek imaging-culture dishes were subjected to laser injury as described previously. Twenty minutes or 6 hours after injury, cells were fixed with 4% paraformaldehyde and permeabilized with ice-cold methanol and blocked in Dulbecco’s PBS/5% goat serum (Dako, X0907)/1% BSA/0.3% Triton X-100 and stained with primary antibody against human LAMP2 (DSHB H4B4; 0.8 μg/ml). Samples were incubated with appropriate Alexa Fluor 568–conjugated antibody (Invitrogen, A10037; 1:1000), and images were taken with the inverted microscope Eclipse Ti-E (Nikon) paired with the UltraVIEW VoX Spinning Disk (PerkinElmer) using the 63× objective.
6
Immunofluorescence Analysis of Cytoskeleton in HTR-8/SVneo Cells
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HTR-8/SVneo cells were pressed on glass slides and fixed in 4% paraformaldehyde for 15 min. The cells were permeabilized with 0.3% Triton X 100 for 10 min and blocked in a 1% bovine serum albumin solution for 1 h. α-Tubulin antibodies (Proteintech, 11224–1-AP) were added and incubated overnight at 4°C. After washing 3 times in PBS with 0.1% Tween-20, the cells were incubated with an Alexa Fluor 568-conjugated antibody (A-11001, Invitrogen) mixture for 1 h at room temperature. Nuclei were stained with DAPI (Solarbio, 28718–90-3, S10 μg/ml) for 15 min, and the cells were washed 3 times before being mounted on glass slides. Microanalysis was conducted using a Leica Stellaris 5 confocal microscope.
Cytoskeleton staining was performed by fixing the cells and staining them with Actin-Tracker Green (C2201S, Beyotime) for 30 min at room temperature in the dark.
Cytoskeleton staining was performed by fixing the cells and staining them with Actin-Tracker Green (C2201S, Beyotime) for 30 min at room temperature in the dark.
7
PTEN Immunofluorescence Staining
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24 h after transfection, cells were fixed using 4% paraformaldehyde in PBS. Cells were permeabilized with 0.1% triton x-100 in PBS, blocked with 10% BSA, and incubated overnight with rabbit anti-PTEN antibody (138G6, Cell Signaling Technology). Coverslips were then incubated with mouse anti-rabbit Alexa Fluor 568-conjugated antibody (Invitrogen), followed by DAPI, and mounted using ProLong Gold antifade mountant (Thermo Fisher Scientific).
8
PTEN Overexpression Analysis by IF
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PTEN expression vectors were generated as previously described [19] (link). Transfection was carried out 24 hours after seeding 50,000 cells in a 12-well dish containing 22x22mm glass coverslips (Thermo Fisher Scientific) using Lipofectamine 2000 (Thermo Fisher Scientific) according to manufacturer's protocols. Successful transfection was confirmed by direct visualization of GFP expression using a fluorescent microscope.
Immunofluorescence 24 hours after transfection, cells were fixed using 4% paraformaldehyde in PBS. Cells were permeabilized with 0.1% triton x-100 in PBS, blocked with 10% BSA, and incubated overnight with rabbit PTEN antibody (138G6, Cell Signaling Technology). Coverslips were then incubated with mouse anti-rabbit Alexa Fluor 568-conjugated antibody (Invitrogen), followed by DAPI, and mounted using ProLong Gold antifade mountant (Thermo Fisher Scientific).
Immunofluorescence 24 hours after transfection, cells were fixed using 4% paraformaldehyde in PBS. Cells were permeabilized with 0.1% triton x-100 in PBS, blocked with 10% BSA, and incubated overnight with rabbit PTEN antibody (138G6, Cell Signaling Technology). Coverslips were then incubated with mouse anti-rabbit Alexa Fluor 568-conjugated antibody (Invitrogen), followed by DAPI, and mounted using ProLong Gold antifade mountant (Thermo Fisher Scientific).
9
Visualizing Actin Dynamics in H. pylori Infection
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AGS cells were seeded on coverslips at 1 × 105 cells/ml and incubated for 24 h at 37°C in 5% CO2. Cells were then serum-starved overnight prior to co-culture with H. pylori for 6 h. Immunofluorescence staining of cellular actin using Alexa Fluor® 488 phalloidin (1:40 dilution; Molecular Probes) was performed as described previously (Hutton et al., 2010 (link)). For staining of bacteria, co-cultured cells or H. pylori that had been air dried and fixed onto microscope slides (Polysine™, Menzel-Glaser, Braunschweig, Germany) were incubated with rabbit anti-H. pylori sera (diluted 1:5,000 in PBS) for 1 h at room temperature, followed by incubation with an anti-rabbit Alexa Fluor® 568 conjugated antibody (1:1,000 dilution; Molecular Probes). The stained bacteria were examined using a Nikon C1 confocal microscope. Actin staining in cells was viewed using the Cellomics ArrayScan VTI HCS Reader (Thermo Scientific), capturing 20 fields per well with the 20 x objective.
10
Visualization of RyR3 Expression
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Stable HeLa clone expressing erGAP3 (see below) were seeded on 12 mm coverslips and transiently transfected with RyR3 cDNA. Cells were fixed for 24-36h with 4% PFA in phosphate buffered saline (PBS) for 20 min; 10% normal goat serum was added for blocking nonspecific binding sites. Expression of RyR3 was detected by incubating the mouse anti-RyR1 antibody (1:200; ThermoFisher) diluted in PBS and containing 10% goat serum, overnight at 4ºC. After washing with PBS, the secondary Alexa Fluor 568conjugated antibody (1:200; Molecular Probes) was added and incubated for 60 min at 22ºC. Cultures were washed with PBS three times and mounted in Vectashield (Vector).
GAP was detected as green fluorescence (excited at 470/40 nm and filtered at 540/50 nm) in a Zeiss Axioplan Z microscope equipped with a 63x/1.2w Korr objective and an AxioCam MR camera. The red fluorescence was excited at 560/40 and filtered at 605/50 nm). The Zeiss ApoTome R system was used for optical sectioning and images were analysed with AxioVision and Image J software.
GAP was detected as green fluorescence (excited at 470/40 nm and filtered at 540/50 nm) in a Zeiss Axioplan Z microscope equipped with a 63x/1.2w Korr objective and an AxioCam MR camera. The red fluorescence was excited at 560/40 and filtered at 605/50 nm). The Zeiss ApoTome R system was used for optical sectioning and images were analysed with AxioVision and Image J software.
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