Streptavidin fitc

HEK293T cells were trypsinized, washed once with FACS buffer (0.75% FCS, 2 mm EDTA) and stained with PE anti-human TNF (MAb11, Biolegend, 1:70) and CaptureSelect^TM biotin anti-C-tag conjugate (Thermo Fisher Scientific, 1:185) for 30 min on ice. After washing in FACS buffer, samples were stained with FITC-streptavidin (Biolegend) for 30 min on ice (1:100) to detect the anti-C-tag conjugate. Cells were subjected to a further wash before analysis on a BD LSR Fortessa. Flow cytometry data were analyzed using FlowJo 10.7. All histograms are shown normalized to mode.

Only samples with typical LP histology were included in the study. After unfreezing, cryosections were incubated with 4% formaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 15 min at 4°C. After double washing with PBS (pH 7.4) they were warmed in microwave in 1 M citrate buffer (pH = 6.0) for 10 min, permeabilised on a shaker in 1% solution of Triton X-100 in PBS, blocked with 2% BSA, and incubated with antibodies against human IL-31 (anti-human biotinylated IL-31 antibody, dilution 1 : 50; Mabtech) overnight at 4°C. Next, slides were thoroughly washed in PBS and incubated with FITC Streptavidin (dilution 1 : 200; BioLegend) for one hour in dark chamber. After another washing, stained slides were mounted with mounting medium (Ultra Cruz Mounting Medium, Santa Cruz Biotechnology) and examined using Zeiss Axio Imager.A2 microscope (Zeiss, Germany). For negative controls, the primary anti-IL-31 antibody was replaced by 0.9% NaCl. The intensity of fluorescence was assessed according to following scale: 0: no reaction, 1: doubtful, 2: week positive, 3: moderately positive, and 4: strongly positive immunoreactivity. All experiments were repeated three times and the mean value for fluorescence intensity was used for further analysis.

Cells were fixed with 4% formalin after 7 days of chondrogenic differentiation or after 1 or 3 days of subsequent osteogenic differentiation. Cells were blocked with 4% bovine serum albumin (Sigma) and immunofluorescent staining was performed using the primary antibodies rabbit anti-Col2a1 (Rockland, 1:100) and goat anti-Col1a1 (Santa Cruz, 1:100) overnight at 4 °C and secondary antibodies AF594 donkey anti-rabbit (Invitrogen, 1:250) and biotinylated donkey anti-goat antibody (Santa Cruz, 1:200) for one hour at room temperature (RT), respectively, followed by incubation with FITC-Streptavidin (Biolegend, 1:200) for 30 min at RT. Host-specific IgG isotype controls were used as a negative control instead of primary antibodies.