Vectastain elite reagent

Mouse femur and tibia were fixed in 10% neutral-buffered formalin, decalcified in 12% EDTA and embedded in paraffin. Sections (5 μm) were stained with hemotoxylin & eosin to evaluate morphology. For 4-HNE immunohistochemistry, bone sections were de-paraffinized, heated in antigen unmasking solution (Dako, Carpinteria, CA) and permeabilized in phosphate-buffered saline (PBS, pH 7.4) containing 0.2% Triton-X-100. Sections were blocked in 1% bovine serum albumin (BSA) for 1 h, incubated with primary antibody (Alpha Diagnostics, San Antonio, TX) overnight at 4°C, followed by incubation in peroxidase-conjugated antibody (Vectastain Elite Reagent, Vector Labs, Burlingame, CA) and in Sigma Fast 3,3′-diaminobenzidine substrate (Sigma, St. Louis, MO). Sections were counterstained with 0.1% Methyl Green in PBS and examined by light microscopy. For quantification, three sections per mouse (n = 3/group) were immunostained twice and analyzed using Image J. Briefly, channels were separated, the blue channel image was inverted and the threshold adjusted to remove nuclear background. Average staining intensity and stained cell counts were measured in several ROI of the same size.

For immunohistochemistry, synovial tissues, RA (n = 12), and OA (n = 8) (patient characteristics are shown in Table S1) or synovial micromass cultures were fixed with paraformaldehyde and embedded in paraffin. Paraffin sections were treated with Tris-EDTA (pH 9). After blocking with goat serum, sections were incubated with primary antibodies (anti-BAFF [TNFSF13B, Enzo Life Sciences] and mTOR or p-MTOR [S2448, both Cell Signaling Technology]) or non-immune immunoglobulins of the same isotype and concentration as the primary antibody (isotype control, anti-rat immunoglobulin [Ig] M [Invitrogen/Thermo Fisher Scientific], and anti-rabbit IgG [R&D]). After incubation with a biotinylated goat anti-rabbit antibody (Vector), sections were incubated with Vectastain Elite reagent and visualized using 3,3-diaminobenzidine (Vector). Sections were counterstained with hematoxylin (Merck). Pictures were taken with an Axioskop 2 microscope (Zeiss) equipped with a digital camera (Olympus).

Synovial tissue samples (patient characteristics are shown in Supplementary Table 1) were fixed in paraformaldehyde and then embedded in paraffin. Paraffin-embedded sections were treated with Tris-EDTA (pH 9). To reduce nonspecific protein binding, the sections were incubated with goat serum. Synovial IRF1 expression was detected with a polyclonal rabbit anti-IRF1 antibody (Cell Signaling Technology). A nonimmune immunoglobulin of the same isotype and concentration as the primary antibody (anti-rabbit IgG (R&D Systems)) served as a control. After incubation with a biotinylated goat anti-rabbit antibody (Vector), the sections were incubated with Vectastain Elite reagent and visualized using 3,3-diaminobenzidine (Vector). The sections were counterstained with hematoxylin (Merck). The expression of IRF1 was assessed using semiquantitative scoring (0 = no staining, 3 = high staining).
IHC was also performed on hind paw tissue from 15-week-old hTNFtg mice and wild-type (WT) littermates. Hind paws were fixed in 7% formaldehyde for 6 h, followed by decalcification in 14% EDTA buffer (pH 7.2) for 4–6 days. Paraffin-embedded sections were used for immunohistochemical staining for synovial IRF1 expression using the antibodies and protocol mentioned above.