Alexa 488 conjugated donkey anti rabbit igg

We confirmed that, unlike a mouse rheumatoid arthritis (RA) model [37 (link)], immunostaining of TNF and IL6 was not increased in DMM surgery (Panels A and B in S2 Fig). Similarly, 10 mM LiCl had no substantial effect on expressions of TNF and IL6 in OAC cells (Panel C in S2 Fig). Sections with the narrowest interarticular space at the medial and lateral compartments were immunostained for β-catenin. First, the paraffin-embedded sections were deparaffinized and rehydrated. Then, the sections were unmasked by 10 mM sodium citrate buffer (pH 6.0) at 90°C for 15 min and were incubated with 3% H₂O₂ for 15 min to inactivate endogenous peroxidases. Sections were blocked with 5% goat serum in TBS-T for 1 hour at room temperature followed by overnight incubation with antibodies against β-catenin (dilution 1:100), TNF, or IL6 (dilution 1: 100 in 5% goat serum with TBS-T, #9587, CST) at 4°C. After washing with TBS-T, sections were incubated with Alexa 488-conjugated donkey anti-rabbit IgG (dilution 1: 500, Thermo Fisher Scientific) for 1 hour at room temperature. Finally, the specimens were mounted in VectaShield containing 2 μg/ml diamidino-2-phenylindole (DAPI, Vector Laboratories) and visualized using the IX71 (Olympus) microscope.

REH cells (3,5x 10⁴ cells per slide) were adhered to poly-L-lysine coated microscopic slides by cytocentrifugation at 370 x g for 4 min. The cells on slides were fixed in 4% PFA for 15 min at room temperature. For analyses of LC3 puncta, the cells were permeabilized with saponin (0.05%) followed by blocking in 2% FBS/PBS for 30 min. The cells were then incubated with antibody against LC3B (PM036, MBL) over-night at 4°C, followed by incubation with Alexa488-conjugated donkey anti-rabbit IgG (A-21206, Thermo Fisher Scientific) for 1 hour at room temperature. Subcellular localization of p53 was determined by incubating the slides over-night with anti-p53 (FL-393Santa Cruz), followed by incubation for 1 hour at room temperature with Cy-3 AffiniPure goat anti-rabbit IgG from Jackson Immunoresearch. For visualization of nuclei, the cells were stained with Hoechst 33258 (1 μg/ml in PBS) from Sigma Aldrich. Fiji was used for quantifying the p53 signals.