Cells grown on coverslips were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized in 0.1% Triton X-100/PBS for 5 min, and then blocked with 5% bovine serum albumin (BSA) in PBS containing 0.05% Triton X-100 for 1 h. Cells were incubated with appropriate primary and fluorophore-conjugated secondary antibodies for 2 h at room temperature (RT), washed, and then mounted with ProLong Antifade (Sigma). For measuring the cell surface level of VEGFR2, VEGFR2 was stained in nonpermeabilized cells, using PBS blocking solution without having Triton in it. Cells were incubated with VEGFR2 primary antibody and fluorophore-conjugated secondary antibodies for 2 h at RT in the blocking buffer having no Triton, washed, and then mounted with ProLong Antifade (Sigma). Images were obtained via a Nikon A1R confocal microscope or Zeiss Axio7/Apatome2 inverted fluorescence microscope with a monochrome camera.
Prolong antifade
Manufactured by Merck Group
ProLong Antifade is a reagent used in microscopy applications to retard the fading of fluorescent signals in fixed samples. It is designed to preserve the intensity and stability of fluorescent dyes and proteins during imaging.
Automatically generated - may contain errors
5 protocols using prolong antifade
1
Immunofluorescence Staining for VEGFR2 Localization
2
Immunofluorescence Staining Protocol for Key Mitotic Proteins
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Immunofluorescence was conducted as previously described[49 (link)]. For Mad2 and BubR1 staining, cells plated on coverslips were pre-extracted with 0.2% Triton X-100 in PHEM (60 mM PIPES (pH 6.8), 25 mM HEPES, 10 mM EGTA and 2 mM MgCl2) for 45 s before fixation with 4% paraformaldehyde in PBS. After staining experiments for Aurora A, Aurora B, H3T3ph, H3S10ph, Bub1 and H2AT120ph, cells were fixed directly in 4% paraformaldehyde before extraction. Then, cells were blocked with 1% bovine serum albumin in TBST for 30 min, incubated with primary antibody for 2 h at room temperature, washed with TBST three times and incubated with secondary antibodies for an additional 1 h at room temperature. DNA was stained with 4,6-diamidino-2-phenylindole for 2–3 min. Coverslips were mounted using ProLong antifade (Sigma). Images were acquired on a DeltaVision microscope (GE Healthcare, Buckinghamshire, UK) with a 60× objective lens, NA=1.42, with optical sections acquired 0.2-0.3 μm apart in the z-axis, and were shown as maximal intensity projections. One exposure setting is used within each experiment. Images shown in the same panel have been identically scaled.
3
Duolink Assay for TGF-β Receptor Interactions
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The Duolink assay (Olink Bioscience) was performed according to the manufacturer’s protocol. Briefly, MDA-MB-231 cells were pretreated with breast cancer conditioned media or 200 ng/ml recombinant fibulin-3, and treated with 50 pM TGF-β1 for 10min. Cells were then washed with PBS, fixed with 4% paraformaldehyde, permeabilized in 0.1% Triton X-100/PBS for 5 min and then blocked with 5% bovine serum albumin in PBS for 1 h. Slides were incubated with anti-TβRI, and anti-TβRII (Santa Cruz Biotechnology, Inc) primary antibodies for 1hrs in room temperature and then incubated with PLA probe MINUS and PLUS mixture for 1h at 37°C. After ligation for 30min, and amplification for 100min at 37°C, the slides were labeled with DAPI, mounted with Prolong Anti-Fade (Sigma), digitally imaged and counted for number of the red dots per cells manually using ImageJ software.
4
Duolink Assay for TGF-β Receptor Interactions
5
Immunofluorescence Staining of Cells
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Cells grown on coverslip that were washed with PBS, fixed with 4% paraformaldehyde, permeabilized in 0.1% Triton X-100/PBS for 5 min, and then blocked with 5% bovine serum albumin in PBS containing 0.05% Triton X-100 for 1 h. Cells were incubated with appropriate primary and fluorophore-conjugated secondary antibodies for 1 h at RT, washed, and then mounted with ProLong Antifade (Sigma).
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