Alexa fluor 546 phalloidin

The spheroids were collected and fixed in 4% paraformaldehyde (PFA). After fixation, the samples were permeabilized with 0.1% triton X-100 in PBS for 30 min and washed with PBS 3 times. For immunostaining, non-specific binding during staining was blocked with PBS containing 4% bovine serum albumin at room temperature for 1 h. Then, we added the anti-SOST antibody (Sigma, USA) and incubated at room temperature for 1 h. After washing with PBS 3 times, the samples were treated with the Alexa Flour 488 secondary antibody (Invitrogen, USA), Alexa Fluor 546 Phalloidin (Invitrogen, USA), and DAPI (Sigma, USA) at room temperature for 1 h. After washing with PBS 3 times, the samples were observed by the FLUOVIEW FV3000 (Olympus, Japan). For G-actin (globular) and F-actin (fibrous) staining, the samples were treated with the Deoxyribonuclease I (DNase I), Alexa Fluor 488 conjugate (Invitrogen, USA) and Alexa Fluor 546 Phalloidin (Invitrogen, USA), respectively, and counterstained with DAPI (Sigma, USA) at room temperature for 15 min. After washing with PBS 3 times, the stained samples were mounted on the glass slides for visualization.

HPV-KER cells were grown on glass sides, fixed with 2% paraformaldehyde (PFA) for 5 min, permeabilized with 0.1% Triton X, 2% PFA-containing phosphate-buffered saline (PBS), and blocked for 2 h at room temperature with PBS containing 1% bovine serum albumin (BSA), 0.05% Triton X 100, and 10% goat serum. Cells were stained overnight at 4°C with anti-human TNIP1 antibody (Sigma Aldrich, St. Louis, MO, United States) or rabbit IgG for isotype control. As a secondary antibody, Alexa Fluor 488 conjugated anti-rabbit IgG, was used for 2 h. Filamentous actin was stained by Alexa Fluor 546® phalloidin (Life Technologies, Carlsbad, United States) diluted 1:100 in PBS containing 1% BSA for 20 min. Nuclei were stained for 10 min with 4′,6-diamidino-2-phenylindole (DAPI) diluted 1:500.
Frozen sections of ex vivo skin models were pre-incubated with PBS for 5 min and fixed and permeabilized with Foxp3 staining buffer set (Thermo Scientific, Rockford, United States) and blocked for 1 h at room temperature with TBS containing 1% BSA and 1% normal goat serum. Cells were stained for 1 h with anti-human TNIP1 antibody or rabbit IgG for isotype control. As a secondary antibody, Alexa Fluor 546 conjugated anti-rabbit IgG (Thermo Scientific, Rockford, United States), was used for 1 h at room temperature. Nuclei were stained for 6 min with DAPI diluted 1:100.

The rat anti-occludin monoclonal antibody (mAb), rabbit anti-occludin polyclonal antibody (pAb) [17 (link)], rat anti-angulin-1/LSR mAb, rabbit anti-angulin-2/ILDR1 pAb [9 (link)], and rat anti-tricellulin mAb (24–69) [7 (link)] were characterized as described previously. The rabbit anti-cleaved-Caspase-3 (Asp175) pAb (# 9661) was purchased from Cell Signaling Technology (Danvers, MA, USA). Alexa Fluor 546-phalloidin (#A22283), Alexa Fluor 488 goat anti-rabbit IgG pAb (#A11008) and Alexa Fluor 546 goat anti-rat IgG pAb (#A11081) were purchased from Life Technologies (Waltham, MA, USA).