SOST, a Wnt inhibitor, was not present in HMWTg knees; however, vector mice had SOST expression in the subchondral bone. partly ameliorate OA abnormalities in subchondral bone and reduce degradative/hypertrophic chondrogenic marker expression in HMWTg joints (15), and Wnt/studies used male homozygous vector and HMWTg without antibody treatment and female homozygous vector and HMWTg mice for antibody treatment studies. Both male and female 5-day-old pups of vector, HMWTg, and LMWTg were used for all studies. Histology Following dissection, knees were fixed in 4% paraformaldehyde, decalcified using a 14% EDTA solution, processed for paraffin embedding in a frontal orientation, and cut into 7-m alternate sections. The ImmunoCruz Rabbit Polyclonal to NOM1 ABC Staining System (Santa Cruz Biotechnology, Dallas, TX) was used for all immunohistochemical staining. After sections were deparaffinized and rehydrated, antigen retrieval was done by incubating sections with 10mM sodium citrate buffer for 10 minutes at 95C. To reduce endogenous target, slides were blocked with 3% hydrogen peroxide in water for 15 minutes, then blocked for 1 hour with 10% serum, and incubated with primary antibodies in blocking buffer at 4C overnight. The following primary antibodies were used: goat anti-sclerostin (SOST) (1:100; R&D Systems, Minneapolis, MN; AF1589; RRID: AB_2195345), rabbit anti-pLRP6 (1:100; Bioss, Woburn, MA; bs-3253R; RRID: AB_10858068), rabbit anti-pLRP5 (1:100; Abcam, Cambridge, MA; ab203306; RRID: AB_2721918), goat anti-WNT5A (1:50; R&D Systems; AF645: RRID: AB_2288488), rabbit anti-WNT7B (1:100; GeneTex, Irvine, CA; GTX114881; RRID: AB_11172941), rabbit anti-pGSK3(1:50; Abcam; ab75745; RRID: AB_1310290), rabbit antiClymphoid enhancer binding factor 1 (LEF1; 1:50; Sigma-Aldrich, St. Louis, MO; AV32404; RRID: AB_1852782), rabbit antiCnon-phospho studies, following removal of skin and bulk muscle of the knee, total RNA was extracted from the entire joint, which included all components above the growth plate D-erythro-Sphingosine using TRIzol reagent (Invitrogen, Waltham, MA) (20). For real-time quantitative reverse transcription PCR analysis, the RNA to cDNA EcoDry Premix Kit (Clontech Inc., Takara Bio, Mountain View, CA) was used to reverse transcribe the RNA to cDNA. iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA) and a MyiQ instrument (Bio-Rad Laboratories) were used for quantitative PCR (qPCR). The relative change in mRNA level was normalized to the mRNA level of studies, RNA was extracted from culture plates using TRIzol reagent and qPCR performed as described above. Additionally, mRNA was normalized to the microcomputed tomography (CT40; ScanCo Medical D-erythro-Sphingosine AG, Bassersdorf, Switzerland). The following morphometric parameters of the region of subchondral trabecular bone of the epiphysis in the femur and tibia were analyzed by three-dimensional (3D) microcomputed tomography: bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp), and trabecular number (Tb.N). FGF23 neutralizing antibody treatment Female HMWTg mice were treated with a rat anti-rat FGF23 neutralizing antibody (FGF23Ab) (Amgen, Thousand Oaks, CA), at a dose of 10 mg/kg body weight or control IgG (rat-anti-NGFPb-3F8-raIgG2a) by intraperitoneal injection twice a week, beginning at 8 weeks of age for 6 weeks. This dosage was previously established in our laboratory in a pilot experiment. Initially, vector mice were treated with control IgG (vehicle; Amgen) only, but an additional experiment that included vector mice treated with FGF23Ab (as an added control group) or control IgG treatment was performed by initiating injections twice a week at 3 weeks of age for 6 weeks and combined into the study. Vector + vehicle, HMWTg + vehicle, and HMWTg + FGF23Ab groups were n = D-erythro-Sphingosine 4, and the vector + vehicle group was n = 3. Mice were euthanized by CO2, and knees were collected. Sections were obtained by methods described, and Safranin-O staining using 0.1% aqueous Safranin-O with 0.02% aqueous Fast Green and Weigert Iron Hematoxylin Counterstain was performed. Chondrocyte culture Chondrocytes were dissected from 5-day-old male and female vector, HMWTg, and LMWTg pups from the femoral head, femoral condyles, and tibial plateau using a dissecting Zeiss microscope. Cartilage was collected in high-glucose DMEM (Life Technologies, Carlsbad, CA) and digested in 0.25% trypsin (Life Technologies) for 30 minutes and then incubated with 500 U/mL of collagenase type II (Worthington Biochemical Corporation, Lakewood, NJ) for 3 hours at 37C. Cells were collected and cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Life Technologies) (22).The cells were plated at a density of 100,000 cells/cm2, incubated for 7 days with media changes every other day, fixed, and stained with alcian blue (3% in acetic acid, pH 2.5) (Poly Scientific, Bay Shore, NY) or Vector Blue Alkaline Phosphatase Substrate (Vector Laboratories). Images were captured with a Nikon TS100 microscope interfaced.