Biotinylated pna

FITC-labeled anti-mouse CD3 (145-2C11, #11-0031-82, 1:100) and GL7 (GL7, #13-5902-81, 1:200) were purchased from eBiosciences (San Diego, CA). Rat anti-mouse B220 (RA3-6B2, #550286, 1:100) and rat anti-mouse IgD (11-26c.2a, #553438, 1:100) were purchased from BD Biosciences (San Jose, USA). Biotinylated PNA (#B-1075,1:300), goat anti-mouse IgM (#FI-2020,1:100) and AMCA-labeled streptavidin (#SA-5008,1:300), biotinylated ulex europaeus agglutinin-1 (UEA-1, #B1065, 1:200) and FITC-labeled streptavidin (#SA-5001, 1:100) were purchased from Vector Laboratories (Burlingame, USA). Texas red-labeled goat anti-rat IgG (#T-6392, 1:500), Alexa Fluor^® 488-labeled goat anti-rat IgG (#A-11006, 1:500), and Alexa Fluor^® 488-labeled goat anti-mouse IgG (#A11001, 1:200) were purchased from Thermo Fisher Scientific (Waltham, USA). Rat anti-mouse cytokeratin 8 (#Troma-1 1:100) was purchased from Developmental Studies Hybridoma Bank (Iowa City, USA).

An in-house ELISA method was set-up and validated for measuring lubricin concentrations in SF, adapted from others^{54 (link),55 (link)}. Monoclonal antibody 9G3 (1 μg/mL in PBS) was coated on 96-well Nunc-Immuno maxisorp plates (Thermo Fisher Scientific) at 4 °C over-night. After blocking with 3% BSA in PBS + 0.05% Tween, SF samples were added as a dilution series (1/50) in assay buffer (1% BSA in PBS-Tween) and incubated for 1 hour at room temperature (RT). Bound proteins were then incubated with biotinylated PNA (Vector laboratories) (1 μg/mL, 1 hour at RT), followed by HRP-streptavidin (Vector laboratories) at 0.1 μg/mL (1 hour at RT). Between each incubation, the wells were washed three times with PBS-Tween to remove unbound reagents. Proteins were stained with 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) until blue colour was fully generated and the reaction stopped by adding 1 M H₂SO₄. Absorbances were read at 450 nm, and compared with a standard curve using recombinant lubricin (dilution series of rhPRG4 (1 mg/mL) in assay buffer). Samples were measured in duplicates and mean values are reported. The lubricin ELISA had an intra plate CV = 7.5% (n = 1 SF sample with 10 repeats) and a inter plate CV% = 7.8% (n = 1 SF sample, tested on 4 plates). Technical performance of the assay is summarized in Supplementary Table 3.

The spleen of immunized mice was separated on day 7 after the final immunization and embedded in optimal cutting temperature (OCT) compound (SAKURA). The tissues were frozen with liquid nitrogen before sectioning (7 µm) on a cryostat. After being fixed in cold acetone and blocked with 5% FBS in PBS at RT for 1 h, the sections were incubated with Biotinylated PNA (VECTOR, 1:100) overnight at 4°C. DyLight 488 Streptavidin (BioLegend, 1:100) was used as the secondary antibody at RT for 1 h followed with Alexa Fluor647-conjugated anti-mouse CD45R (BioLegend, 1:150) at RT for 1 h. After staining, the sections were scanned under a Pannoramic SCAN instrument (3DHISTECH, Hungary).