Human bronchial epithelial cells (HBECs, Lonza/Fischer Scientific, Göteborg, Sweden) were seeded on poly-L lysine-coated glass coverslips, placed in a 24-well plate, and maintained in bronchial epithelium cell medium (BEpiCM, ScienCell, Carlsbad, CA, USA) in a 5% CO2 incubator at 37 °C until 80–90% confluence. After washing and fixation in 4% paraformaldehyde, cells were permeabilized using Triton X-100 (0.1% in phosphate-buffered saline, PBS). This was followed by washing, blocking with 5% bovine serum albumin (BSA) in PBS with Tween® 20 (PBST), and labeling with a murine monoclonal antibody against p63 (1:250; ab735, Abcam, Cambridge, UK). This was visualized after incubation at room temperature (RT) for 1 h with an Alexa Fluor 594-conjugated goat anti-mouse secondary antibody (1:500; Thermo Fischer Scientific, Waltham, MA, USA). A primary murine monoclonal antibody against MUC5AC was used (1:250; MA1-38223, Invitrogen, Carlsbad, CA, USA) and visualized using the method described for detection of p63. Nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI; Prolong Gold antifade reagent with DAPI, Thermo Fisher Scientific).
Alexa fluor 594 conjugated goat anti mouse secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor 594-conjugated goat anti-mouse secondary antibody is a fluorescently labeled antibody that binds to mouse primary antibodies. It is designed for use in immunoassays and other applications that require the detection of mouse-derived targets.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Lsm 700, by Zeiss (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Bepicm, by ScienCell (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Ma1 38223, by Thermo Fisher Scientific (1 mentions)
Ab735, by Abcam (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Hif 1α, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Sp8 inverted confocal laser scanning microscope, by Leica (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Goat serum, by Merck Group (1 mentions)
20 protocols using alexa fluor 594 conjugated goat anti mouse secondary antibody
1
Immunofluorescence Staining of HBECs
2
Immunofluorescence Staining of ASTC-a-1 Cells
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ASTC-a-1 cells were seeded into 6-well plates for 24h before treatments. The cells were fixed for 20 min at room temperature, and then permeabilization was applied with ice-cold methanol at -20° C for 10 minutes. The cells were washed with PBS twice after permeabilization and incubated in blocking buffer (5% normal goat serum) for 60 min at room temperature, followed by incubation with the primary antibodies: HIF-1α (Cell Signaling Technology, cat# 36169), β-catenin (Cell Signaling Technology, cat# 8814), vimentin (Cell Signaling Technology, cat# 5741), Slug (Cell Signaling Technology, cat# 9585), E-Cadherin (Cell Signaling Technology, cat# 3195) and β-actin (Santa Cruz Biotechnology, cat# sc-47778) (diluted 1:50 in blocking buffer) at 4° C overnight. After incubation, the cells were washed with PBS three times, followed by incubation with Alexa Fluor 594-conjugated goat anti-mouse secondary antibody (Thermo Fisher, cat# A-11005) and Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Thermo Fisher, cat# A-11034). Finally, the nuclei were labeled with DAPI (Sigma, cat# D9542) before observation. The images were analyzed with a confocal microscopy system (LSM700, Zeiss).
3
Immunofluorescence Analysis of Myc-AIP Localization
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MEFs were cultured overnight on glass coverslips in six-well plates. After transfection and/or virus infection, cells were washed with PBS, fixed with paraformaldehyde (ThermoFisher Scientific) for 15 min, and washed three times with PBS. Cells were then permeabilized with 0.5% Triton-X in PBS for 5 min, followed by three PBS washes. Cells were blocked in 3% bovine serum albumin for 1 h. MEFs transfected with Myc-AIP plasmids were stained with mouse anti-Myc antibody (1:1000 in 3% bovine serum albumin) overnight. On the following day, cells were washed three times with PBS before incubation with Alexa Fluor 594–conjugated goat anti-mouse secondary antibody (1:1000, ThermoFisher Scientific). Coverslips were washed six times with PBS and then mounted onto slides using ProLong Diamond Antifade Mountant with DAPI staining (ThermoFisher Scientific). Images were obtained with a Leica SP8 Inverted Laser Scanning Confocal Microscope using a 63× oil objective. Images were analyzed using Fiji and JaCOP plugin for colocalization analysis. Nuclear translocation was quantified by analyzing the ratio of GFP in the nucleus to GFP fluorescence in the entire cell using Fiji.
4
Cellular Distribution Analysis of Influenza Nucleoprotein
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For cellular distribution studies, 3D printed constructs were fixed in 4% formaldehyde (Sigma) and permeabilised with 1% Triton-X-100 for 15 min. Subsequently, nuclear staining was performed with 1 µg/ml Hoechst (H33342, AppliChem, Germany) for 1 h at room temperature.
For immunofluorescence staining, constructs were fixed and permeabilised as described above. Afterwards, constructs were blocked with 5% goat serum (Sigma) for 30 min and incubated with diluted anti-influenza NP antibody (ab20343, Abcam, UK, 1:1,000) overnight at 4 °C. Afterward, constructs were incubated with Alexa Fluor 594-conjugated goat-anti-mouse secondary antibody (A11005, Thermo Fisher Scientific, USA; 1:2,000) overnight at 4 °C. Nuclear counterstaining was performed using Hoechst stain (1 µg/ml) for 1 h at room temperature. Cellular distribution and immunofluorescence were analysed with the Zeiss Observer. Z1 microscope (Zeiss, Germany).
For immunofluorescence staining, constructs were fixed and permeabilised as described above. Afterwards, constructs were blocked with 5% goat serum (Sigma) for 30 min and incubated with diluted anti-influenza NP antibody (ab20343, Abcam, UK, 1:1,000) overnight at 4 °C. Afterward, constructs were incubated with Alexa Fluor 594-conjugated goat-anti-mouse secondary antibody (A11005, Thermo Fisher Scientific, USA; 1:2,000) overnight at 4 °C. Nuclear counterstaining was performed using Hoechst stain (1 µg/ml) for 1 h at room temperature. Cellular distribution and immunofluorescence were analysed with the Zeiss Observer. Z1 microscope (Zeiss, Germany).
5
Zebrafish Ciliary Dynamics Imaging
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Zebrafish control and injected embryos were fixed at 8–12 ss, for KV analysis, in 4% PFA (Sigma) for 24 h at 4 °C, then washed in 1X PBS- 1% Triton X-100 (PBST; Sigma) and dehydrated gradually through a methanol series and stored in 100% methanol (Sigma) at −20 °C. Whole-mount immunofluorescence experiments were performed as follows: embryos were first washed with PBST several times, blocked with goat serum 5% for 1 h before incubation with primary and secondary antibodies both in PBS-Triton (1%) plus 2% goat serum overnight at 4 °C. We stained KV cilia using a mouse anti-acetylated α-tubulin (1:750; T7451, Sigma) monoclonal antibody and, subsequently, with Alexa Fluor 594-conjugated goat anti-mouse secondary antibody (1:1000, ThermoFisher). Nuclei were stained with DAPI.
Finally, embryos were mounted in 75% glycerol and confocal images were acquired on a Leica SP5 microscope and analysed using ImageJ software. Identical settings (gain, offset, laser power) were applied to all z-stack acquisitions, and all fluorescence measurements were performed on maximum intensity projection, also calculated with ImageJ.
Finally, embryos were mounted in 75% glycerol and confocal images were acquired on a Leica SP5 microscope and analysed using ImageJ software. Identical settings (gain, offset, laser power) were applied to all z-stack acquisitions, and all fluorescence measurements were performed on maximum intensity projection, also calculated with ImageJ.
6
Immunofluorescence Assay for Ki67 and β-Actin
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HCT116 cells were seeded into 6-well cell culture plates and incubated overnight to establish adherence. The cells were fixed with 3.7% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization in ice-cold methanol for 15 minutes at -20o C. Subsequently, the cells were incubated with blocking buffer (PBS containing 5% normal goat serum and 0.5% Triton X-100) for 1 hour at room temperature, followed by incubation with Ki67 (cat# ab209897, Abcam) and β-actin (cat# sc-47778, Santa Cruz Biotechnology) antibody (diluted 1:500 in blocking buffer) at 4° C overnight. Next, the cells were washed three with PBS for 10 minutes, and then incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (cat# A-11034; Thermo Fisher) for Ki67 and Alexa Fluor 594-conjugated goat anti-mouse secondary antibody (cat# A-11004; Thermo Fisher) for β-actin (diluted 1:500 in blocking buffer) and at room temperature. Nuclei were stained with DAPI (cat# D9542; Sigma) for 30 minutes at room temperature before observation. The Nikon Eclipse E800 fluorescence microscope was used to capture the images.
7
Immunostaining of KLF5 and β-actin in A549 cells
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A549 cells were seeded into 6‐well plates and allowed to adhere for 24 h before treatments. The cells were fixed for 10 min at room temperature and then permeabilised with ice‐cold methanol at −20°C for 15 min. After permeabilisation, the cells were washed twice with PBS and blocked for 60 min at room temperature in a blocking buffer (5% normal goat serum). The primary antibodies, including KLF5 (Cell Signalling Technology, cat# 40674) and β‐actin (Santa Cruz Biotechnology, cat# sc‐47778) were diluted 1:100 in blocking buffer and incubated with the cells overnight at 4°C. After incubation with the primary antibodies, the cells were washed three times with PBS and then incubated with Alexa Fluor 594‐conjugated goat anti‐mouse secondary antibody (Thermo Fisher, cat# A‐11005) and Alexa Fluor 488‐conjugated goat anti‐rabbit secondary antibody (Thermo Fisher, cat# A‐11034). Before imaging, the nuclei were stained with DAPI (Sigma, cat# D9542) for 30 min. The images were acquired using a confocal microscopy system (LSM700, Zeiss) and analysed.
8
Protein Transfection and Immunofluorescence Imaging
9
Multicolor Immunofluorescence Staining
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Using a PBS solution containing 4% paraformaldehyde, the cells were fixed on 48well plate slides. The cells were then permeated with PBS containing 0.3% Triton-X 100 for 15 min and blocked for 1 h at room temperature with PBS containing 10% sheep serum. After adding the primary antibody and allowing it to incubate overnight, the cells were washed three times with PBS and then incubated with the corresponding secondary antibody: donkey anti-rat IgG (H+L) highly cross-adsorbed secondary antibody (CD68(1:500, Abcam, Waltham, MA, USA, ab31630), F4/80(1:200, Abcam, Waltham, MA, USA, ab6640), and RABGGTB (1:500, GeneTeX, San-Antonio, TX, USA, GTX105874)), Alexa Fluor 488 (1:1000; Invitrogen, Carlsbad, CA, USA; A21208), Alexa Fluor 594-conjugated goat anti-mouse secondary antibody (1:1000; Thermo Fisher Scientific, Waltham, MA, USA; #A-11032), and goat anti-rabbit secondary antibody labeled with Alexa Fluor 647 (1:1000, Invitrogen, A21245). After 1 h of staining at room temperature with 4 ,6-diamidino-2phenylindole (DAPI), the cell nuclei were visible. The cells were then washed three times with PBS and observed using a confocal fluorescence microscope (ZEISS, Oberkochen, Germany; LSM900). The parameters of the microscope were set at the start of each individual imaging process and remained constant throughout.
10
Spinal Cord Tissue Analysis after Injury
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Seven weeks after spinal cord injury, rats were deeply anesthetized using 10% chloral hydrate, perfused transcardially with ice-cold heparinized (10 U/mL) saline, and then injected with 4% PFA in PBS. The spinal cord was dissected, and fixed in 4% PFA solution in 0.1 M PBS for 48 h at 4°C. The 8-mm-long spinal cord tissue centered on the lesion was frozen and sliced in 20 μm thick. In brief, the sections were rehydrated in PBS for 5 min followed by incubation with 1% bovine serum albumin and 0.01% triton-X100 for 1 h at room temperature. Then the sections were exposed to appropriate primary monoclonal antibodies (1:300 Tuj-1, Abcam; 1:300 GFAP Abcam; 1:500 MBP; 1:200 Synaptophosin) in a humidified chamber at 4 °C overnight. After washing three times with PBS, the slides were then incubated with an Alexa Fluor 594-conjugated goat anti-mouse secondary antibody (1:800, Molecular Probes) or Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:400, Molecular Probes) at 4 °C in the dark for 2 h. Finally, the sections were mounted onto glass slides for microscopic analysis after staining with 4',6-Diamidino-2-phenylindole (DAPI) and following extensive rinsing with PBS. The cellular and morphological structures of the spinal cord tissue were assessed by H&E staining.
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