Tritc conjugated donkey anti mouse igg

Briefly, cells were fixed and permeabilized using, respectively, 4% paraformaldehyde in PBS and 0.2 M NH₄Cl/PBS containing 0.2% Triton X-100 for 10 min at room temperature. After blocking with 3% bovine serum albumin (BSA; Sigma-Aldrich) in PBS for 2 h, F-actin and the nuclei were stained with Phalloidin-TRITC (2 μg/mL; Sigma-Aldrich) and DAPI (1 μg/mL; Sigma-Aldrich) for 60 min at room temperature, respectively. To characterize Calu-3 spheroid polarity, cells were first incubated with rabbit polyclonal anti-ZO-1 (1:100 dilution; Zymed Laboratories Inc.) and mouse monoclonal anti-integrin β1 antibodies (1:500 dilution; Abcam) overnight at 4°C. To identify apoptosis within Calu-3 spheroids, cells were first incubated with rabbit polyclonal active Caspase-3 (1:200 dilution; Abcam) overnight at 4°C. After washing with PBS 3 times (5 min each), the cells were incubated with FITC-conjugated donkey anti-rabbit IgG and TRITC-conjugated donkey anti-mouse IgG (both 1:200 dilution; Jackson ImmunoResearch) for 1 h to detect ZO-1, active Caspase-3 and β1 integrin, respectively. Samples were lastly washed 3 times with PBS, followed by examination under a confocal microscope (LSM710, Zeiss, Germany).

The following primary Abs were used: Rabbit anti‐Fam70a Ab (Cat#: bs‐11006R; Bioss); Rabbit anti‐Wnt5a Ab (Cat#: A12744; ABclonal); Rabbit anti‐adenomatous polyposis coli (APC) Ab (Cat#: AF0547; Affinity Biosciences); Rabbit anti‐Phospho Catenin Beta (Ser33/37/Thr41) Ab (Cat#: DF2989; Affinity Biosciences); Rabbit anti‐Phospho Akt (Thr308) Ab (Cat#: 13038; Cell Signaling Technology); Rabbit anti‐Phospho Akt (Ser473) Ab (Cat#: A5030; Bimake.Com); Mouse anti‐β‐actin Ab (Cat#: A5316‐100); Mouse anti‐β‐tubulin Ab (Cat#: sc‐5274); Mouse anti‐GAPDH Ab (Cat#: 30201ES60; YEASEN); Rabbit anti‐Beta‐Catenin Ab (Cat#: A5038; Bimake.Com); Mouse anti‐Strep II Tag Ab (Cat#: YFMA0054; YI FEI XUE BIOTECHNOLOGY); Mouse anti‐Flag Tag Ab (Cat#: YFMA0036; YI FEI XUE BIOTECHNOLOGY).
The following secondary Abs were used: TRITC‐conjugated Donkey anti‐Mouse IgG (Code: 715‐025‐150) and Cy2‐conjugated Donkey anti‐Rabbit IgG (Code: 711‐225‐152) were purchased from Jackson ImmunoResearch Laboratory. Horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit IgG and HRP‐conjugated goat anti‐mouse IgG were purchased from Vazyme. Donkey Anti‐rabbit IgG/Gold (35 nm, Cat#: bs‐0295D‐Gold) and Donkey Anti‐rabbit IgG/Gold (15 nm, Cat#: bs‐0295D‐Gold) were purchased from Bioss.