Cells were grown on glass dishes for 24 h then fixed for 15 min in 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 for 15 min at room temperature. Next, cells were blocked with 10% goat serum for 1 h at room temperature, followed by overnight incubation with primary antibodies at 4°C. After being washed with PBS, the cells were incubated with Alexa-conjugated secondary antibody (Cell Signaling Technology, Inc.) at 37°C for 1 h and then incubated with DAPI at 37°C for 5 min. The cells were visualized and images captured on a confocal microscope (Olympus LV3000; Olympus Corporation). Primary antibodies were: RPL9 (dilution: 1:50; Abcam; cat. no. ab182556), TSG101 (dilution: 3:100; MilliporeSigma; cat. no. SAB2702167), VPS4A (dilution: 1:50; Santa Cruz Biotechnology; cat. no. SC-393428) and Alix (dilution: 1:50; Santa Cruz Biotechnology; cat. no. sc-53540).
Alexa conjugated secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Alexa-conjugated secondary antibodies are fluorescently labeled antibodies that bind to primary antibodies. They are used in various immunodetection techniques, such as immunofluorescence microscopy and flow cytometry, to enable visualization and quantification of target proteins or cells.
Automatically generated - may contain errors
Lab products found in correlation
Lv3000, by Olympus (2 mentions)
Vps4a, by Santa Cruz Biotechnology (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Trypsin, by Corning (1 mentions)
Phospho gsk3β, by Cell Signaling Technology (1 mentions)
Eif4e, by Cell Signaling Technology (1 mentions)
Phospho cyclin d1, by Cell Signaling Technology (1 mentions)
Phospho gsk3α, by Cell Signaling Technology (1 mentions)
Pnu 74654, by Santa Cruz Biotechnology (1 mentions)
Mg132, by Merck Group (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
6 protocols using alexa conjugated secondary antibody
1
Immunofluorescence Staining of Exosome Markers
2
Immunofluorescence Staining of Cultured Cells
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Cells were grown on glass dishes for 24 h, and then treated with 0.3% Triton X-100 for 15 min after being fixed in 4% paraformaldehyde for 15 min at room temperature. After that, 10% goat serum in PBS was used to block the cells for 60 min at room temperature. After the incubation with primary antibodies and Alexa-conjugated secondary antibody (Cell Signaling Technology, USA), the cells were stained with DAPI for 5 min and visualized on a confocal microscope (Olympus LV3000, Japan).
3
Immunofluorescence Staining of ARF6 and DUSP6
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Cancer cells were seeded on culture slides at low density and cultured to 60%–70% confluency. For immune-staining, cells were washed twice with PBS, and fixed with 4% paraformaldehyde for 20–30 min at 4°C. Permeabilization was performed with 1% Triton-X100 for 5–15 min at room temperature after being washed twice with PBS. After wash with PBS, the slides were blocked with 1% BSA for 30 min. The antibodies used were anti-ARF6 and anti-DUSP6 (1:100 dilution) and were shown to be specific. Slides were incubated with primary antibody at 4°C overnight. After being washed with PBS, slides were incubated with Alexa-conjugated secondary antibodies (1:2000 dilution; Cell Signaling Technology) for 30 min at room temperature. Cells were then washed with PBS and the nuclei were stained with DAPI. Finally, the slides were observed under a fluorescence microscope (Leica).
4
Flow Cytometry of Cell Markers
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Cultured cells were first dissociated with trypsin (Corning) from plates into single-cell suspension. For intracellular marker detection, cells were fixed, permeabilized and blocked. The cells were then incubated with primary antibody for one hour. For surface marker detection, cells were blocked and then incubated with primary antibodies for one hour. After primary antibody incubation, Alexa-conjugated secondary antibodies (Cell Signaling Technologies) were then added to cell suspension for thirty minutes before FACS detection. The GPR68 antibody (sc-98437) was obtained from Santa Cruz. The αSMA antibody (ab119952) was obtained from Abcam. For analysis of subpopulation in tumours, cells were first dissociated from tumours using 0.25% trypsin for 30min and 0.1% collagenase for 24 hours into single cell suspension.
5
Wnt/β-catenin Inhibitors and Their Effects
6
Proliferating Cell Nuclear Antigen Labeling
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Proliferating cell nuclear antigen (PCNA) can be used to label proliferating cells [26 (link)]. Double-staining immunofluorescence was carried out to determine the LC proliferation rate as previously described [17 (link)]. The slides were permeabilized with 0.1% Triton X-100. After primary antibody incubation, Alexa-conjugated secondary antibodies (Cell Signaling Technology, Boston, MA, USA) were followed to label CYP11A1 (green) and PCNA (red). The PCNA-labeling index was presented.
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