Phosphate buffered saline (pbs)

Cells were fixed in 4% paraformaldehyde/PBS for 15 min at room temperature and permeabilized with 1% Triton X-100/PBS (Sigma) for 10 min. After blocking with 10% donkey serum in PBS (Jackson ImmunoResearch) for 1 h at room temperature, cells were incubated with primary antibodies overnight at 4 °C, followed by incubation with appropriate fluorescent-conjugated secondary antibodies for 1 h at room temperature the next day. Primary antibodies were as follows: OCT4 (Abcam), SOX2 (Abcam), SSEA4 (Abcam), TFAP2C (Santa Cruz), PRDM14 (Abcam), and NANOS3 (Abcam). Secondary antibodies were as follows: AlexaFluor 488 conjugated donkey anti-rabbit IgG and AlexaFluor 594 conjugated donkey anti-mouse IgG (all Life Technologies). The nuclei were counterstained with DAPI (Thermo Fisher Scientific). The cells were observed with a Zeiss inverted confocal microscope.

Cells seeded on coverslips (Thermo Fisher Scientific) were washed twice in PBS and fixed in 4% paraformaldehyde (PFA) for 30 min. 0.4% Triton X-100 (Sigma) was diluted by PBS, in which cells were permeabilized for 30 min. Then, cells were blocked in 10% donkey serum in PBS (Jackson ImmunoResearch) for 1 h (RT). After incubation with primary antibodies in blocking buffer at 4 °C overnight, cells were incubated with secondary antibodies at room temperature for 1 h and labeled with Hoechst33342 (Thermo Fisher Scientific) to visualize nuclei. Images were taken with a Leica SP5 confocal microscope.

Mouse paw tissue paraffin embedded sections from 7 month old COMP deficient 129/Sv mice^{32 (link)} and age matched WT mice were analysed. Paraffin removal and then antigen retrieval were obtained by incubation in an alkaline solution (K8004 EnVision FLEX Target Retrieval Solution, High pH 9.0, DAKO, Denmark A/S, Glostrup, Denmark) at 85 °C for 40 min. Sections were cooled in washing buffer (K8007 EnVision FLEX Wash Buffer, DAKO) and blocked for 1 h at room temperature (RT) with 2% normal donkey serum in PBS (Jackson ImmunoResearch Laboratories, INC., West Grove, PA), also used for antibody dilution. Primary rabbit anti-lubricin antibody (P3-118, Thermo Scientific) at 2 µg/ml was incubated overnight at 4 °C followed by incubation with the secondary antibody for 1 h at RT with 3 × 5 min intermediary washes with washing buffer. The secondary antibody was either Rhodamine Red-X-conjugated, donkey anti-rabbit (711-296-162, Jackson ImmunoResearch, diluted 1 in 1000), or biotin-conjugated, donkey anti-rabbit (Jackson ImmunoResearch, diluted 1 in 1000) visualised after incubation with streptavidin-FITC for 30 min at RT. Negative control was performed with primary antibody omitted. Finally, sections were mounted as above and images were captured in a Zeiss Axioscope 2 Plus fluorescence microscope and images were cropped and contrast adjusted for the complete image in Adobe Photoshop CC.

Cells seeded on coverslips (Fisher) were fixed in 4% paraformaldehyde (PFA) for 15 min, permeabilized with 0.4% Triton X-100 in PBS for 10 min, and blocked with 10% donkey serum in PBS (Jackson ImmunoResearch) for 1 h. Cells were then incubated with primary antibody in the blocking buffer at 4 °C overnight, washed with PBS three times, incubated with secondary antibody at room temperature for 1 h, washed again with PBS three times, and counterstained with Hoechst 33342 (Invitrogen). Quantitative microscopy was performed using randomly chosen cells for each sample^{15 (link)}. The number, intensity, and area of fluorescent signals were analyzed using Image J software (NIH).

After we allowed ARPE-19 cells to grow in high-glucose medium (25 mM) for three days, the cells were fixed with cold methanol, blocked with 25% nonimmune goat serum (Sigma-Aldrich), incubated with the mouse antihuman NF-κB p65 antibody (1:100 diluted in PBS) covering the cell surface for 1 h at 37 °C, and then washed three times with PBS. Subsequently, fluorescein isothiocyanate-conjugated goat antimouse IgG (1:200 dilution) was applied for 1 h. The samples were washed three times with PBS (Jackson ImmunoResearch, West Grove, PA) and placed in a blocking solution at 25 °C for 3 h. All steps were performed according to the manufacturer’s instructions. Nuclei were counterstained with DAPI. The fluorescence intensity was measured using a fluorescent microplate reader (Bio-Rad Laboratories), and related images were captured.