Immpress universal reagent kit

IHC was performed using anti-RLN2 (LS-B10495; LifeSpan BioSciences, Seattle, WA, USA), anti-RXFP1 (CB-18419; Proteintech, Rosemont, IL, USA), anti-RXFP2 (NLS4753; Novus Biologicals, Littleton, CO, USA), and anti-periostin antibodies (ab14041; Abcam, Cambridge, UK). Specimens were then incubated with anti-mouse/rabbit IgG antibodies provided with the ImmPRESS UNIVERSAL REAGENT kit (Vector Laboratories, Burlingame, CA, USA). The reaction was developed with Histofine Simple Stain DAB (Nichirei Biosciences, Tokyo, Japan) and counterstained with methyl green or hematoxylin. For fluorescent immunostaining, we used anti-MMP-1 (aa169–464, FITC, LS-C418498C418498; LifeSpan), anti-iNOS (2072R-FITC; Bioss, MA, USA), and anti-sclerostin (SOST) antibodies (AF1589, R&D Systems). Secondary antibodies for SOST were Alexa Fluor 488 rabbit anti-goat IgG (H + L) (A-11078; Invitrogen). Samples were mounted with ProLong Diamond Antifade Mount with DAPI (Thermo Fisher Scientific, MA, USA). DAB-positive areas were measured using NIH image software.

Immunocytochemical analysis was performed using an ImmPRESS Universal Reagent kit (Vector Laboratories, Inc.). hOCs or hOBs (1×10⁶/cm² and 1×10⁴/cm² cells, respectively) were seeded in 24-well plates, fixed in cold 100% methanol at room temperature for 10 min and permeabilized with 0.2% (v/v) Triton X-100 in TBS (1X). Then, the cells were treated in 0.3% H₂O₂ in TBS (1X) for 10 min at room temperature, and subsequently incubated with ready-to-use (2.5%) normal horse serum blocking solution (ImmPRESS Universal Reagent kit) for 15 min at room temperature.
After the incubation in blocking serum, cells were incubated at 4°C overnight following addition of the following rabbit anti-human polyclonal primary antibodies: Runx2 (1:200); COL1a1 (1:100); NFATc1 (1:300); cathepsin K (1:200); and OPN (1:200). After rinsing in 1X TBS, the cells were incubated for 30 min at room temperature with ImmPRESS reagent and then stained with substrate/chromogen mix (ImmPACT™ DAB). After washing, the cells were mounted in glycerol/PBS (9:1), counterstained with hematoxylin and observed with a Nikon Eclipse 50i optical microscope (magnification, ×20; Nikon Corporation).