Alexa fluor 594 conjugated secondary antibody

MDC staining was used to detect the formation of acidic vesicular organelles (AVOs) in breast cancer cells. Briefly, MCF7 and MDA-MB-231 cells were seeded on the 24-well plates and treated with 1/4x, 1/2x, 1x, 2x IC₅₀ of SAHA or 1x IC₅₀ of resveratrol for 72 h. AVOs were labeled with 0.5 mM MDC in the phenol red-free RPMI at 37°C for 2 h. Then, the cells were washed three times with PBS. To detect the formation of autophagosome or autophagolysosome in cells, immunofluorescence staining of LC3B was used. Briefly, MCF7 cells were seeded on glass coverslips. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature and the fixed cells were then permeabilized with PBS containing 1% triton X-100 (Calbiochem) for 30 min, subsequently blocked in 5% bovine serum albumin (Sigma-Aldrich) for an hour and incubated with anti-LC3B primary antibodies overnight at 4°C. The cells were washed three times with TBS-Tween buffer, incubated with Alexa Fluor 594-conjugated secondary antibodies (Abcam, cat# ab150076) for an hour at room temperature, and suspended in blocking buffer containing DAPI for 15 min. AVOs and autophagosome/autophagolysosome in all cells were observed under a fluorescence microscope (Olympus, IX-71). Experiments were repeated at least three times.

IF analysis was performed as described [31 (link)]. Cells grown on cover-glasses were fixed in 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked with 5% bovine serum. Then the cells were incubated with antibodies against Ki67 (1:500, Abcam, USA) overnight at 4°C, followed by Alexa Fluor 594-conjugated secondary antibodies (1:500, Abcam, USA). Nuclei were dyed by DAPI and images were observed under a fluorescence microscope (Leica, Germany).

To detect intracellular influenza viral proteins, cells were permeabilized using a Foxp3/transcription factor staining kit (Tonbo Biosciences) according to the manufacturer's instructions. Briefly, after fixation and permeabilization of the cells, anti‐influenza viral M1 (5 µg/mL, Abcam) or NP antibodies (2.5 µg/mL, Abcam) were added, followed by staining with Alexa fluor 488‐conjugated secondary antibodies (10 µg/mL, Thermo Scientific) or Alexa fluor 594‐conjugated secondary antibodies (10 µg/mL, Abcam). Data were collected using an Attune™ NxT Acoustic Focusing Cytometer (Thermo Scientific) and analysed using a FlowJo software (BD Biosciences).⁴³