NCI-H295 cells (2x105) were seeded on coverslips in 24-well plates 24 hours before siRNA transfection. Forty-eight hours after transfection, the cells were fixed in methanol and blocked with 10% normal horse serum. Beta-catenin was detected using a monoclonal mouse primary anti-B-Catenin antibody (dilution: 1:2000; #610154, BD Biosciences) and a goat anti-mouse IgG1 FITC secondary antibody (dilution: 1:250; sc_2078, Santa Cruz Biotechnology). Nuclear staining was made using DAPi (dilution: 1:25000 #4083, Cell Signaling Technology) and slides were set with Fluoromount (Sigma-Aldrich). Fluorescence was acquired with an Imager.A1 fluorescence microscope (Zeiss) using the AxioVision LE software (Zeiss) with fixed exposure time for all samples.
Imager a1 fluorescence microscope
Manufactured by Zeiss
Sourced in Germany
The IMAGER A1 is a fluorescence microscope designed for high-resolution imaging. It is capable of capturing detailed images of fluorescently labeled samples. The microscope utilizes advanced optics and illumination systems to provide clear and precise visualization of cellular and subcellular structures.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Fluoromount, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Cell Signaling Technology (1 mentions)
Axiovision le software, by Zeiss (1 mentions)
Rhodamine phalloidin, by Merck Group (1 mentions)
Facs software, by BD (1 mentions)
Axiovert lsm 710, by Zeiss (1 mentions)
Alexa fluor 488 ab, by Thermo Fisher Scientific (1 mentions)
Alexa 488 or alexa 568 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Clone rpa t4, by BioLegend (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Edta antigen retrieval solution, by Wuhan Servicebio Technology (1 mentions)
Sox2 pe, by BD (1 mentions)
Perm buffer, by BD (1 mentions)
Vectashield antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
Axiovision, by Zeiss (1 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
12 protocols using imager a1 fluorescence microscope
1
Immunofluorescence Analysis of Beta-Catenin
2
Immunohistochemical Analysis of CD4 and CD103
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Paraffin-embedded samples were cut into 5 μm sections, and processed for immunohistochemistry as previously described (Wu et al., 2018b (link)). Briefly, tissue sections were fixed with 4% paraformaldehyde, followed by membrane permeabilization using 0.2% Triton-X-100. Then, the coverslips were incubated in 5% BSA, and were sequentially incubated with primary CD4 (Clone T4, Biolegend) or CD103 (Ber-ACT8, Biolegend) antibody and secondary Alexa Fluor® 488 secondary Ab (Thermo, Product # A-11034) or Alexa Fluor® 594 secondary Ab (Thermo, Product # A-11005) before mounting. Finally, the coverslips were observed under a ZEISS IMAGER A1 fluorescence microscope (CARL ZEISS) to capture fluorescence images.
3
Cellular Uptake of Gold Nanoparticles by Wharton's Jelly MSCs
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The Wharton’s jelly MSCs (1 × 104 cells/well) were seeded on 24-well culture plates with 15 mm glass coverslips for overnight incubation. After cell attachment, the MSCs were incubated with AuNP-Col-FITC (Control: no treatment; AuNP: 1.25 ppm, 2.5 ppm, 5 ppm, 10 ppm) for 30 min, 2 h, and 24 h at 37 °C with 5% CO2 atmosphere. After the incubation, the cells were washed with PBS solution three times, fixed with 4% paraformaldehyde (PFA) for 30 min, washed thrice with PBS, and 0.5% Triton X-100 (Sigma-Aldrich, USA) was added for permeability for 10 min. The FBS solution was added into each sample for blocking (4 °C, 30 min). Next, the cell skeleton (F-actin) was stained by 1:400 dilution of Rhodamine phalloidin (Sigma-Aldrich) for 1 h in the dark, while the cell nuclei were stained by 4,6-diamidino-2-phenylindole (DAPI, 1 mg/mL, Invitrogen, USA) solution for 10 min and washed thrice by PBS solution. Finally, the fluorescence was observed using a Zeiss Axio Imager A1 fluorescence microscope and analyzed by Image J 5.0 software. The fluorescein-positive cells were also detected with a flow cytometer to analyze cell uptake efficiency by fluorescence-activated cell sorting (FACS) software (BD Biosciences, USA). The experiments were executed in triplicate.
4
LC3 Autophagy Visualization in Macrophages
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THP-1 macrophages and RAW264.7 cells were seeded on the coverslips pre-coated with collagens in 24-well plates. After transfection with siRNA for 24 h or addition with the recombinant BD2 and BD3 (1 µg/ml) for 6 h, cells were infected with PA at a multiplicity of infection (MOI) 1 for 6 h. Next, cells were fixed with 4% paraformaldehyde, followed by membrane permeabilization using 0.2% Triton-X-100. Then, the coverslips were incubated in 5% BSA, and were sequentially incubated with primary LC3 antibody and secondary Alexa Fluor 488 goat anti-rabbit IgG Ab before mounting. Finally, the coverslips were observed under a ZEISS IMAGER A1 fluorescence microscope (CARL ZEISS) to capture fluorescence images. For confocal microscopy, cells were treated with siRNA or BD2/3 and DQ-red BSA (lysosome marker) for 6 h, and thereafter challenged with Zymosan Alexa Fluor 488 Fluorescent Bioparticles at MOI 25 for 1 h. After fixation and blockade, nuclei were then stained with the blue-fluorescent dye DAPI, and cells were visualized using a confocal microscope (Zeiss Axiovert, LSM710).
5
Immunohistochemical Analysis of T-cell Markers
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Paraffin-embedded samples were cut into 5-μm sections, and processed for immunohistochemistry as previously described (54 (link)). Briefly, tissues were fixed with 4% paraformaldehyde, followed by membrane permeabilization using 0.2% Triton-X-100. The coverslips were then incubated in 5% BSA, and were sequentially incubated with TCRa7.2 (Clone REA179, Miltenyi) and IL-9 (American Research Products, Product #E-AB-27215) or OX40 (Clone Ber-ACT35, Biolegend) Abs, followed by secondary Alexa Fluor® 488 Ab (Thermo, Product # A-11034) or Alexa Fluor® 594 Ab (Thermo, Product # A-11005) and DAPI (Thermo, Product # A-11034) staining before detection. Finally, the coverslips were observed under a ZEISS IMAGER A1 fluorescence microscope (CARL ZEISS) to capture fluorescence images.
6
Quantifying Phytophthora Infection in Hairy Roots
7
Immunofluorescence Analysis of Lung Tissue
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Fresh lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 4-μm sections. After deparaffinating, rehydration and retrieval, tissues were incubated with primary antibodies overnight at 4°C. After rinsing several times with PBS, sections were incubated with Alexa-488- or Alexa-568-conjugated secondary antibodies (Invitrogen, United Kingdom) for 2 hours. Finally, DAPI was added onto slides for 10 minutes. Images were obtained with a ZEISS Imager A1 fluorescence microscope (Gottingen, Germany). Immunofluorescence was performed using the following primary antibodies: rabbit monoclonal anti-P21 and anti-PTEN antibodies (Abcam, United Kingdom); and mouse monoclonal anti-SPC antibody (Santa Cruz Biotechnology, CA, USA).
8
Immunohistochemical Analysis of CD4 and IL-21
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Paraffin-embedded samples were cut into 5-μm slices, and immunohistochemistry was conducted as previously described (4 (link)). In brief, the mucosa was fixed with paraformaldehyde, followed by antigen retrieval with the EDTA antigen retrieval solution (Servicebio). The processed samples were blocked with 5% bovine serum albumin (BSA) and subsequently incubated with CD4 (Clone RPA-T4, BioLegend) and IL-21 (Abbiotec, Product #253397) Abs, followed by staining of secondary Abs Alexa Fluor® 488 (Thermo, Product #A-11034) and Alexa Fluor® 594 (Thermo, Product #A-11005) as well as DAPI (Thermo, Product #A-11034) before the detection. Fluorescence images were captured under a ZEISS IMAGER A1 fluorescence microscope (CARL ZEISS).
9
Stem Cell Marker Expression Analysis
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Cells were fixed using 4% v/v paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA), washed three times with PBS containing 1% BSA and permeablized using Perm Buffer (BD Biosciences, San Jose, CA, USA) for 15 min at room temperature (Intracellular markers). After permeabilization, cells were blocked with PBS containing 1% BSA for 1 h at room temperature after the blocking solution was removed and cells were washed three times with PBS. Cells were then incubated with conjugated antibodies in PBS containing 1% BSA overnight at 4 °C. Antibodies used were at 1:50 dilution factor for Oct4-PE (BD Biosciences, San Jose, CA, USA), Sox2-PE (BD Biosciences, San Jose, CA, USA) Nanog-ALexa Fluor 488 (BD Biosciences, San Jose, CA, USA), and Tra-1-60-PE (BD Biosciences, San Jose, CA, USA). After the overnight incubation, cells were washed three times with PBS and stained with DAPI ANTIFADE GOLD (Invitrogen, Carlsbad, CA, USA) prior to viewing under Zeiss Imager A.1 Fluorescence Microscope (Carl Zeiss, Oberkochen, Germany).
10
Immunofluorescence Staining of Cell Lines
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Raji, HL60, and Jurkat cell lines and normal lymphocytes were stained with FITC-conjugated antibodies, namely isotype control, anti-NaK antibody, or anti-TK1 antibody (CB1) for 30 minutes on ice and in the dark. Cells were washed with cold DPBS and then were resuspended at 5×105 cells/mL. Approximately 20 µL of cell solution was placed on a glass slide, after which a drop of mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) was added to the sample (Vectashield Antifade Mounting Medium with DAPI; Vectashield, Burlingame, CA, USA), and a coverslip was placed on top. Cells were visualized in a light microscope (Zeiss Imager A.1 Fluorescence Microscope; Carl Zeiss Meditec AG, Jena, Germany) using different channels to detect fluorescence. Blue represents DAPI fluorescence and green represents FITC fluorescence.
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