Rabbit polyclonal igg osteocalcin

Sectioned femur samples (n = 6/group) (pre-treated in xylene for 10 min to clear away the paraffin) were incubated with testicular hyaluronidase for 30 min to expose collagen epitopes. The samples were immunofluorescently labeled for 1 h at room temperature either with rabbit polyclonal IgG collagen type II (10 μg/ml; ab34712; Abcam, UK) followed by Alexa 488 donkey anti rabbit IgG (2 μg/ml; Invitrogen, Eugene, OR, USA) or rabbit polyclonal collagen IX (10 μg/ml; Abcam) followed by Alexa 647 goat anti-rabbit IgG (2 μg/ml; Invitrogen) or Rabbit (Rb) pAb collagen X (10 μg/ml; ab58632; Abcam) followed by Alexa fluor 488 donkey anti-rabbit (2 μg/ml; Invitrogen) or rabbit polyclonal IgG osteocalcin (10 μg/ml; Santa Cruz Biotechnology, CA, USA) followed by Alexa fluor 488 donkey anti-rabbit (2 μg/ml; Invitrogen). Antibodies were diluted in 3% (w/v) bovine serum albumin (BSA). The nuclear stain bisbenzimide (Sigma Aldrich; Hoechst dye No. 33258, dissolved in H₂O) was administered for 5 min and coverslips were mounted on slides using Airvol as described previously [21 (link), 22 (link)]. Images were taken (n = 8 image sections/sample) on the Zeiss 780 confocal with a 20× objective (0.75NA, Beam Splitter (MBS) 458/514/561/633, 5% laser output, and (MBS) 405, 2% laser output). Images were quantified using ImageJ (NIH, Bethesda).

Cells were isolated from three female osteoporotic patients whose ages were 60, 73, and 76. The cells were seeded in a 24-well plate at a density of 1 × 10⁵ cells/cm² per well. Once 90% confluent cells were serum-starved overnight and treated with 100 nm CK2.3 or 40 nm BMP2 or left unstimulated. After five days of treatment, cells were washed with 1X PBS and then fixed with acetone and methanol. The samples were fluorescently labeled for one hour at room temperature for rabbit polyclonal IgG osteocalcin as a 1:200 dilution (200 μg/mL, Santa Cruz Biotechnology, Dallas, TX, USA), which was followed by Alexafluor chicken antirabbit as a 1:500 dilution (200 μg/mL, Life Technologies, Carlsbad, Ca, USA) and goat polyclonal IgG alkaline phosphatase as a 1:200 dilution (200 μg/mL, Santa Cruz Biotechnology), followed by Alexafluor 568 donkey antigoat IgG as a 1:500 dilution (200μg/mL, Life Technologies, Carlsbad, CA, USA). All antibodies were diluted in a 3% bovine serum albumin (BSA) solution. Bisbenzimide (Sigma-Aldrich, St. Louis, MO, USA Hoechst dye No. 33258, dissolved in H₂O) was used as a nuclear stain for two and a half minute incubation. The coverslips were mounted using Airvol, as previously described [37 (link),38 (link)]. Images were taken on Zeiss Axiophot with a 20 × objective (Fluor, Zeiss, Germany) and analyzed in ImageJ (NIH, Bethesda, MD, USA).