Cells were fixed in 4% paraformaldehyde (Merck) in PBS for 10 min at room temperature (RT). Fixed cells were permeabilized with 0.05% Tween 20 (Merck) in PBS. Incubation with the primary antibody against acetylated-α-tubulin (6-11B-1, 1:2000, Sigma-Aldrich) was performed overnight at 4℃. This was followed by 1 h incubation with secondary Cy3-labeled goat-anti-mouse antibody (1:500, Vector Laboratories) and DAPI nuclear counterstaining (1:1000, Molecular Probes) for 5 min at RT. Samples were mounted in Prolong Gold (Molecular Probes).
Dapi nuclear counterstaining
Manufactured by Thermo Fisher Scientific
DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye used for nuclear counterstaining in various microscopy techniques. It binds strongly to DNA, allowing visualization of cell nuclei. DAPI emits blue fluorescence when excited by ultraviolet or violet light.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Lsm 510 confocal laser scanning microscope, by Zeiss (1 mentions)
Cy3 labeled goat anti mouse igg, by Beyotime (1 mentions)
Bovine serum albumin (bsa), by GE Healthcare (1 mentions)
Bovine serum albumin bsa, by GE Healthcare (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
3 protocols using dapi nuclear counterstaining
1
Immunofluorescence Assay for Acetylated Tubulin
2
Immunostaining of ACE2 and PEDV-N
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For immunofluorescence staining, cells were washed twice with PBS, followed by membrane permeabilization with 0.1 % Triton X-100 for 5 min. Fixed cells were incubated with 5 % BSA for 1 h at room temperature. The cells were incubated with mouse monoclonal antibody ACE2 (1:400, Abcam) or PEDV-N (1:100, Medgenel) overnight at 4 °C. After rinsing three times with PBS, CY3-labeled goat anti-mouse IgG (1:1000, Beyotime, China) was added and incubate with the cells for 1 h at room temperature. Nuclei were visualized using DAPI nuclear counterstaining (Molecular Probes). Pictures of immunofluorescent were captured using an LSM 510 laser scanning confocal microscope (Zeiss, Germany).
3
Immunofluorescence Staining of Cells
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For immunofluorescence staining, cells were washed twice with PBS and fixed with 3.7% formaldehyde (Merck, 1040031000) in PBS, followed by membrane permeabilization with 0.1% Triton-X-100 (Sigma, 93426) in PBS for 10 min at room temperature. Fixed cells were incubated with 3% Bovine serum albumin (BSA) (GE Healthcare Life Sciences) in PBS for 1 h followed by incubation with the primary antibody for 1 h in PBS with 1% BSA. After rinsing three times with PBS, staining was completed by Alexa Fluor®488-conjugated goat α rabbit antibody (Life Technologies, A11008) or Alexa Fluor®488-conjugated goat α mouse antibody (Life Technologies, A11001). Nuclei were visualized using DAPI nuclear counterstaining (Molecular Probes). Pictures of immunofluorescent cells were captured using an EVOS-fl fluorescence microscope (Advanced Microscopy Group) at 10 x magnification. Percentage of infected cells (relative to mock-treated) was calculated by counting infected cells in 10 x microscopic fields.
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