Cells from the second passage were seeded and cultured on coverslips precoated with type IV collagen for 24 h. The cultured cells were washed with PBS and fixed with 70% methanol in acetone. After being washed three times with PBS, the coverslips were blocked for 1 h with 1% BSA (Sigma, Saint Louis, USA) at room temperature (RT). Then, the cells were incubated sequentially with mouse anti-human β1 integrin (1:100, Santa Cruz, California, USA) at 4 °C overnight and with TRITC-conjugated donkey anti-mouse secondary antibody (1:500, Invitrogen, California, USA) for 1 h at RT. Next, the cells were stained with rabbit anti-human CK19 (1:100, Sigma, Saint Louis, USA) for 12 h at 4 °C and FITC-conjugated goat anti-rabbit secondary antibody (1:500, Invitrogen) for 1 h at RT. Finally, the stained cells were examined using a laser-scanning confocal fluorescence microscope (Leica, Munich, Germany).
Laser scanning confocal fluorescence microscope
Manufactured by Leica
Sourced in Germany
The Laser Scanning Confocal Fluorescence Microscope is an imaging instrument that uses a focused laser beam to scan a specimen and detect fluorescence emitted from the sample. It provides high-resolution, three-dimensional imaging of fluorescently labeled biological samples.
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6 protocols using laser scanning confocal fluorescence microscope
1
Immunofluorescence Analysis of Integrin and Cytokeratin
2
Immunofluorescence Imaging of Cellular P1
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Cells were treated with P1, fixed, and permeabilized. P1 was conjugated with using click chemistry. Subsequently, the cells were incubated with primary antibodies, secondary fluorescent antibodies, and Hoechst dye. Cellular images were obtained using a laser scanning confocal fluorescence microscope (Leica, Hamburg, Germany). JC-1 (BD Biosciences, Franklin Lake, NJ, USA) staining was also performed.
3
Immunofluorescence Staining of Mouse EpSCs
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Mouse EpSCs were seeded onto coverslips pre‐coated with type IV collagen, fixed with 70% methanol in acetone and blocked for 1 hour with BSA 1% (Sigma) at ambient temperature. The cells were then incubated with rabbit anti‐mouse MMP9 (GTX100458, GeneTex, 1:400) at 4°C overnight, followed by a one‐hour incubation with phycoerythrin (PE)‐labelled donkey anti‐rabbit second‐class antibody (1:500; Invitrogen) under the same conditions and a five‐min incubation with DAPI (5 μg/mL, Sigma). The stained cells were examined under a laser scanning confocal fluorescence microscope (Leica).
4
Visualizing HA-CD44 Binding Specificity
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Cells were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS, Macgene Biotech) and antibiotics (penicillin 100 U/mL and streptomycin 100 mg/mL). All the cells were cultured in incubators maintained at 37°C with 5% CO2 in a humidified atmosphere. MDA-MB-435 cells were seeded on 6-well plates and incubated in complete medium for 24 h at 37°C. Then, the medium was replaced with fresh culture medium containing FITC conjugated HANP/HCPT (FITC-HANP/HCPT) and incubated for 4 h at 37°C. To verify the specificity of HA binding with CD44, free HA (1 mg/mL) was added to cells 30 min before HANP/HCPT. After washing with PBS (pH = 7.4) for three times, cells were fixed in cold ethanol at −20°C for 15 min. After being fixed, cells were labeled with DAPI in darkness for 10 min and then imaged by a laser scanning confocal fluorescence microscope (Leica, German) with specific filter for FITC.
5
Immunocytochemical Analysis of Trop-2, E-cadherin, and Galectin-3
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Wild-type or mutated Trop-2 expressing HCT116 cells were stained immunocytochemically with anti-FLAG-tagged Trop-2, anti-E-cadherin and anti-galectin-3 antibodies as described previously (23 (link)).
When an intracellular antigen was detected, cells were fixed with methanol at −20 °C for 40 min, and then washed with PBS. After permeabilization with PBS containing 1% TX-100 at room temperature for 20 min, the cells were treated with methanol at −20 °C for 40 min. After blocking with PBS containing 5% BSA and 0.1% TX-100, the cells were incubated with primary antibodies (rabbit anti-HA tag and mouse anti-β-catenin antibodies), and then stained with fluorescence-conjugated secondary antibodies and DAPI. Finally, the stained cells were observed under a confocal laser-scanning fluorescence microscope (Leica) at a magnification of ×630.
When an intracellular antigen was detected, cells were fixed with methanol at −20 °C for 40 min, and then washed with PBS. After permeabilization with PBS containing 1% TX-100 at room temperature for 20 min, the cells were treated with methanol at −20 °C for 40 min. After blocking with PBS containing 5% BSA and 0.1% TX-100, the cells were incubated with primary antibodies (rabbit anti-HA tag and mouse anti-β-catenin antibodies), and then stained with fluorescence-conjugated secondary antibodies and DAPI. Finally, the stained cells were observed under a confocal laser-scanning fluorescence microscope (Leica) at a magnification of ×630.
6
Evaluation of Apoptosis in Kidney Tissue
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Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling assay (TUNEL assay) was performed using an in situ cell death detection kit (Roche, Penzberg. Germany) according to the manufacturer's instructions. Briefly, tissue taken from kidneys was fixed and embedded in paraffin and 4-μm sections were prepared. After dewax and rehydrate, sections were stained with terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling solution and incubated at 37 °C for 1 h, washed twice with phosphate-buffered saline, and mounted with Prolong Gold antifade solution containing DAPI. The amount of DNA fragmentation was visualized using a confocal laser scanning fluorescence microscope (Leica, Heidelberg, Germany). Nuclear counterstaining (blue) was performed using DAPI. Positive and negative controls were treated with 0.1 mg/mL pancreatic DNase I or labeling respectively. The number of TUNEL positive nuclei was determined by examining six randomly selected microscopic fields from each experimental group. The staining was analyzed in a blinded fashion.
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