Cells were fixed with 4% paraformaldehyde for 30 min at room temperature and then endogenous peroxidase/phosphatase activity was blocked with 3% H2O2 for 20 min in a dark environment. The cells were then incubated with an anti-α-smooth muscle actin (α-SMA) antibody (1:200, 55135-1-AP; ProteinTech Group, Inc.) at a dilution of 1:1,000 with 5% BSA (Roche) and incubated overnight in a humidified box at 4˚C. Cy3-labeled fluorescent secondary antibody (AS1109; ASPEN Biotechnology) was added at a dilution of 1:100 after washing, and incubated for 50 min at room temperature. Then, the cells were washed three times with 1X PBS, and `````counterstained with DAPI (AS1075; ASPEN Biotechnology) for 5 min at room temperature. Confocal microscopy was performed and fluorescence was analysed using a fluorescence microscope (Leica DMI4000B; Leica Microsystems, Inc.).
As1075
Manufactured by Aspen
Sourced in China
The AS1075 is a laboratory equipment that performs centrifugation. It is designed to separate different components of a liquid sample based on their density and size.
Automatically generated - may contain errors
4 protocols using as1075
1
Immunofluorescence Staining of α-SMA in Cells
2
Immunofluorescence Analysis of HCC Cells
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For IF analysis, cells were washed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 min. HCC cells were then permeabilized with 0.5% NP-40 in PBS for 20 min and blocked with 5% bovine serum albumin for a duration of 30 min. Next, cells were incubated with primary antibodies for 2 h and incubated with secondary antibodies for 1 h. For the nuclei, cells were stained with DAPI (ASPEN, AS1075). Finally, confocal microscopy (OLYMPUS, IX51) was used for image acquisition.
3
Blue Laser Irradiation Effects on Bladder Cancer Cell Proliferation
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Bladder cancer cells (1 × 106) were plated in 6-well chamber slides and exposed to blue laser for 4 J/cm2, 8 J/cm2, or no irradiation. After 48 h, the cells were fixed with 4% paraformaldehyde for 30 min and incubated with blocking solution for 10 min at 20°C for permeabilization and non-specific antigen blocking. Next, the cells were incubated with rabbit anti-Ki67 primary antibody (1:150 dilution, 27309-1-AP, Proteintech, USA) overnight at 4°C. The cells were then incubated with goat anti-rabbit antibody for 50 min (AS-1109, ASPEN Biotechnology, China) as a secondary antibody, and the nuclei were stained with 4′,6-diamidino-2-phenylindole (AS1075, ASPEN Biotechnology, China). Images of five random fields were obtained for each group using a fluorescence microscope (BX51, Olympus, Japan). Ki67-positive cells were stained red in the nucleus. Five random fields were chosen from each slice, and the percentage of Ki67-positive cells was determined.
4
Evaluating Apoptosis in Human ES A673 Cells
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Human ES A673 cells were seeded onto sterile coverslips (10212432C; Citotest Labware Manufacturing Co., Ltd, Jiangsu, China), which were inserted in 6-well plates (5 × 105 cells/well). 48 h after the transfection with 15 nM nCAR/miR-34a-5p, control RNA or vehicle, the coverslips were fixed with 4% paraformaldehyde (AS1018; Aspen, Wuhan, China). After being permeabilized and blocked, the coverslips were incubated with primary antibody against cleaved-caspase-3 (1:200; AF7022; Affbiotech, OH, USA) at 4°C overnight, and then incubated with FITC labeled goat anti-rabbit secondary antibody (1:50; AS-1110; Aspen, Wuhan, China). Nucleus was counterstained with DAPI (AS1075; Aspen, Wuhan, China). Images were taken and observed with a fluorescence microscope (IX51; Olympus Corporation, Shinjuku, Tokyo, Japan). Cleaved-caspase-3-positive cells were counted under the fluorescence microscope for percentages of positive stained cells which were compared between different groups of treatments (38 (link), 39 (link)).
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