8 chamber slide

NHEKs seeded into 8 chamber slides (Thermo Fisher Scientific) were incubated with various combination of U1 RNA, poly(I:C), cathelicidin peptides, and fucoidan for 1 hour at 4 °C allowing binding, but not internalization. Unbound RNA was removed by washing with cold PBS. Cells were fixed with 4% PFA at 4 °C. Blocking buffer (Sigma) was used to prevent nonspecific antibody binding, and cells were incubated with two primary antibodies. Secondary antibodies conjugated with oligonucleotides were added, and hybridization, ligation, amplification and detection steps were performed according to the manufacturer’s instructions (Sigma) to generate an amplified fluorescent signal in areas where the antigens recognized by the two primary antibodies reside within less than 40 nm. Fluorescent PLA signals were evaluated using fluorescence microscopy (described above). Signals are counted by Duolink ImageTool software version 1.0.1.2 (Sigma-aldrich).

The immunofluorescence and immunohistochemical staining were performed on the tissue samples of surgical knee OA as reported previously^{14 (link)}. For immunofluorescence staining, the rehydrated sections were retrieved in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). After the sections were blocked with 2% BSA in PBS for 1 h, they were incubated with the primary antibodies for TG2 (1:200 dilution), β-catenin (1:400), non-p-β-catenin (1:400), FoxO3a (1:200), and p-FoxO3a (1:200) or normal rabbit IgG (1:200) in 1% BSA in PBS at 4 ℃ overnight. For immunohistochemistry, the retrieved sections were immunostained for MMP3 (1:200) and MMP13 (1:400) in 1% BSA in PBS for 16 h at 4 ℃ and then with goat anti-rabbit IgG conjugated with HRP (DakoCytomation, Glostrup, Denmark) for 1 h at RT.
For immunocyto-fluorescence, primary chondrocytes were plated on 8-chamber slides (3 × 10⁴ cells/well, Thermo Fisher Scientific) and treated with TGF-β1 (10 ng/ml), with or without calcium ionophore (2 mM) or ZDON (100 μM). The cells were fixed with 4% formalin for 5 min, permeabilized with 0.25% Triton X-100 in PBS for 3 min, blocked with 5% BSA for 1 h, and incubated with rabbit anti-non-p-β-catenin (1:400) and mouse anti-TG2 (1:200) antibodies at RT for 1 h.

For immunofluorescence staining, cells were cultured on 8-chamber slides (Thermo Fisher Scientific). Cells were treated with 100 nmol/L of each compound for 5 days. After treatment, cells were fixed using 3.6% formaldehyde in PBS for 15 min. The fixed cells were incubated for 20 min in 0.2% Triton-X in PBS at room temperature (RT) for permeabilization followed by incubation with 50 mmol/L NH₄Cl for 15 min and 30 min in 3% BSA in PBS at RT for blocking. Cells were incubated with primary VDR (1:200) antibody (Merck Darmstadt, DE) for 1 h at RT or Ki67 (1:500) (Thermo Fisher Scientific) antibodies for 1 h at RT. An Alexa Fluor 647 conjugated anti-rabbit secondary antibody was used at a concentration of 1:500 in PBS for VDR staining visualization. Next cells were incubated with Ki67 (1:500) antibody (Thermo Fisher Scientific) for 1 h at RT. The Ki67 antibody had a fluorescence marker. Cells were counterstained using DAPI (Thermo Fisher Scientific) and mounted using Fluoromount G (Southern Biotech, Birmingham, AL, USA). Images of the stained cells were acquired using TissueFAXS hard- and software (TissueGnostics GmbH, Vienna, Austria) equipped with a Zeiss AxioImager Z1 using a Zeiss NeoFluar 20×/0.5 objective (Zeiss, Oberkochen, Germany).

Immunofluorescence staining of CCSP-2 was performed by seeding HCT116/CCSP-2 and HCT116/pcDNA cells (30,000 cells/well) in 8-chamber slides (Thermo Fisher Scientific, MA, USA) and fixation with 4% paraformaldehyde. The cells were washed with PBS and blocked with 5% normal goat serum (Cell Signaling Technology, MA, USA) in 1% BSA in PBS-Tween 20 (PBS-T) as the dilution buffer for 60 min. FITC-conjugated anti-CCSP-2 scFv and control scFv (5 μg/mL) in dilution buffer were added to each cell line for 60 min after washing with PBS. PBS-washed cells were incubated with 4,6-diamidino-2-phenylindole (DAPI; Invitrogen, CA, USA) to stain the nucleus for 10 min, and the cells were visualized with a LSM880 confocal microscope (Carl Zeiss, Jena, Germany).

Cells were plated on 8-chamber slides (Thermo Fisher Scientific) at approximately 2 × 10⁴ cells/chamber and incubated overnight at 37°C. Following removal of the medium, cells were fixed in 4% formalin (Sigma-Aldrich) for 15 min at RT, and then treated with 0.1% Triton X-100 in PBS for another 15 min. After washed twice with PBS, cells were incubated with 10 μg/ml of hPAM4 and a murine mAb against MUC5AC, α-MUC1, or a rabbit polyclonal antibody against MUC17 in PBS plus 1% BSA for 45 min at RT. Afterwards, cells were washed twice and incubated with a mixture of FITC-GAH and Cy3-GAM or Cy3-GAR in PBS plus 1% BSA for 30 min at RT. After three washes, chambers were dissembled. Slides were mounted with an antifade solution (VectaShield, Vector Laboratories) containing the nuclear counterstain, 4, 6-diamidino-2-phenylindole (DAPI). Image acquisition and analyses were performed using an Olympus fluorescence microscope with a Kodak camera system.