THP-1 macrophage (2 × 105 cells/ml) were cultured on Lab-Tek chambered cover glasses (Nalgene Nunc International, Rochester, NY), treated with nanoparticles and then were fixed and permeabilized in cold 70% methanol for 30 min at 4°C. Cells were washed in PBS, incubated with Image-iTTM FX signal enhancer (ThermoFisher Scientific) for 30 min at room temperature in a humid environment. Cells were washed in PBS then incubated with primary antibody (polyclonal goat anti-Dectin-1, Santa Cruz Biotechnology, Dallas, TX) overnight at 4°C. Cells were washed with PBS and incubated for 1 hr at room temperature with a secondary antibody conjugated to ALEXA Fluor 647 (Alexa Fluor® 647 donkey anti-goat IgG, ThermoFisher Scientific). Cells were then counterstained with the nuclear stain 4′, 6-diamidino-2-phenylindole dilactate (DAPI, ThermoFisher Scientific) and imaged by confocal microscopy using a Leica Confocal Laser Scanning Microscope (TCS SP2 AOBS, Leica Microsystems, Heidelberg GmbH) with an oil immersion objective lens 63X. An Argon 488 nm laser was applied to excite FITC; Laser operating at 405 nm was applied to excite DAPI [16 ].
Image ittm fx signal enhancer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Image-iTTM FX Signal Enhancer is a laboratory equipment designed to enhance the fluorescence signal in microscopy applications. It is intended to improve the visualization and detection of fluorescent-labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Alexa fluor 647 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Lc3 2, by Cell Signaling Technology (1 mentions)
Zen 2009 light edition software, by Zeiss (1 mentions)
Keap1, by Merck Group (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
F2168, by Merck Group (1 mentions)
P1951, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
5 protocols using image ittm fx signal enhancer
1
Immunofluorescence Staining of Dectin-1 in THP-1 Macrophages
2
Visualizing Rickettsial Autophagy in Cells
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ECs were seeded on glass coverslips and infected with R. rickettsii as described above. At 6h post-infection, cells were washed with PBS, fixed with 3.7% formaldehyde, permeabilized with 0.2% Triton X-100 for 20 min, and incubated with Image-iTTM FX signal enhancer (Thermo Fisher) to block any background staining. The cells were then incubated with a rabbit polyclonal antibody against LC3-II (Cell Signaling) followed by an Alexa Fluor 488 secondary antibody. Cells were also stained with an anti-R. rickettsii antibody followed by a compatible Alexa Fluor 568 secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The staining was observed using a FluoView® FV10i Olympus confocal microscope and software (Center Valley, PA, USA) and images were captured using a camera system connected to the microscope.
3
Porcine Embryo Protein Expression Analysis
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To observe the protein expression during Ge-132 treatment and determine the key signal pathways, porcine embryos were sampled from control (no Ge-132) and 200 μg/ml Ge-132 treatment groups at day 7 after IVF. They were
fixed with 4% paraformaldehyde for 40 min, then permeabilized with 1% Triton X-100 for 30 min. After incubation in Image-iTTM FX Signal Enhancer (I36933, Invitrogen) for 30 min, the embryos were further blocked with 1%
bovine serum albumin (BSA, A9418) in PBS for 1 h. They were then incubated with a primary antibody overnight at 4°C. The primary antibodies used were KEAP1 (ABS97, Millipore, Temecula, CA, 1:200) and BCL2 (SC23960, Santa
Cruz, Dallas, TX, 1:200). After completely washing and secondary blocking, they were further incubated with the appropriate secondary antibodies. After the nuclei were stained with 10 µg/ml Hoechst 33342 (B2261), the
embryos were mounted with anti-fade reagent (S36937, Invitrogen) and observed under laser-scanning confocal microscope (LSM700, Carl Zeiss, Oberkochen, Germany) and ZEN 2009 Light Edition software (Carl Zeiss).
fixed with 4% paraformaldehyde for 40 min, then permeabilized with 1% Triton X-100 for 30 min. After incubation in Image-iTTM FX Signal Enhancer (I36933, Invitrogen) for 30 min, the embryos were further blocked with 1%
bovine serum albumin (BSA, A9418) in PBS for 1 h. They were then incubated with a primary antibody overnight at 4°C. The primary antibodies used were KEAP1 (ABS97, Millipore, Temecula, CA, 1:200) and BCL2 (SC23960, Santa
Cruz, Dallas, TX, 1:200). After completely washing and secondary blocking, they were further incubated with the appropriate secondary antibodies. After the nuclei were stained with 10 µg/ml Hoechst 33342 (B2261), the
embryos were mounted with anti-fade reagent (S36937, Invitrogen) and observed under laser-scanning confocal microscope (LSM700, Carl Zeiss, Oberkochen, Germany) and ZEN 2009 Light Edition software (Carl Zeiss).
4
Dynamics of Cytoskeletal Structures during Polar Body Extrusion
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To observe the microtubule, microfilament and chromosome dynamics during p2PB extrusion, piPSNT embryos were sampled from control (no 6DMAP) treatment and 6DMAP treatment groups at 0, 1.5, 4 and 8 hpa, respectively. They were fixed with 4% paraformaldehyde for 40 min, then permeabilized with 1% Triton X-100 for 30 min. After incubation in Image-iTTM FX Signal Enhancer (I36933, Invitrogen, Carlsbad, CA) for 30 min, the embryos were further blocked with 1% bovine serum albumin (A9418, Sigma, St. Louis, MO) in PBS for 1 h. They were then incubated with TRITC conjugated phalloidin (1:200, P1951, Sigma, St. Louis, MO) at 37°C for 2 h. After completely washing and secondary blocking, they were further incubated in FITC conjugated monoclonal anti-α-tubulin (1:200, F2168, Sigma, St. Louis, MO) for another 3 h. After the nuclei were stained with 10 μg/ml Hoechst 33342 (B2261, Sigma, St. Louis, MO), the embryos were mounted with anti-fade reagent (S36937, Invitrogen, Carlsbad, CA) and observed under laser-scanning confocal microscope (Zeiss, LSM700).
5
Immunofluorescence Imaging of Vinculin
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Samples were washed with phosphate-buffered saline (PBS) three times and fixed with 4% formaldehyde in PBS for 15 min and then permeabilized with 0.25% Triton X-100 in PBS for 10 min. Fixed cells were pre-incubated at room temperature with signal enhancer (Image-iTTM FX signal enhancer, Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) to prevent non-specific background staining before immunostaining with a 1:400 dilution of primary antibody for human vinculin (Sigma Aldrich, Saint Louis, MO, USA) for an hour at room temperature. Alexa 546 goat anti-mouse secondary antibody (Invitrogen, Thermo Fisher Scientific) was treated at a 1:200 dilution for an hour. F-actin was stained with Alexa Fluor 488 phalloidin (Invitrogen, Thermo Fisher Scientific) for 30 min, and the samples were placed on a glass slide with the patterned side touching the surface, followed by nucleus staining and mounting with mounting solution with DAPI (4',6-diamidino-2-phenylindole Vector laboratories, Burlingame, CA, USA) for 15 min. Fluorescence images were taken using an optical microscope. (IX71, Olympus, Shinjuku, Tokyo).
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