Heterogeneous SF-SCs were isolated from the femurs of a 6–8-week-old sheep fetus (obtained at the local abattoir) by flushing with L-15 medium supplemented with Anti-Anti (Thermo Fisher Scientific). Cells were then expanded in vitro and verified for multipotency according to differentiation methods for chondrogenesis61 (link), osteogenesis62 (link), and myogenesis63 (link). Differentiated and undifferentiated SF-SCs were fixed in 4% buffered formaldehyde and stained with a wide range of primary antibodies (1 h at room temperature) from Abcam (Amsterdam, Netherlands), including α-SMA (ab32575; 1:500), vimentin (ab8798; 1:100), CD166 (ab235957; 1:200), Ki67 (ab15580; 1:300), estrogen receptor-α (ER-α; ab66102; 1:100), ER-β (ab187291; 1:100), progesterone receptor (PR; ab2765; 1:100), cytokeratin (ab9377; 1:1000), MyoD1 (ab16148, 1:100), RANK (ab13918, 1:100), and DMP1 (ab103203, 1:100). Each primary antibody was conjugated with either CY3 or Alexa Fluor 488 secondary antibodies (Thermo Fisher; 1:300) and DNA was labeled with DAPI. Chondrogenesis was evaluated after alcian blue staining using standard methods.
Ab13918
Manufactured by Abcam
Sourced in Netherlands
Ab13918 is a primary antibody specifically designed for the detection of a target protein. It is suitable for use in various immunoassay techniques, including Western blotting, immunohistochemistry, and flow cytometry. This product is manufactured by Abcam and is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Ab9377, by Abcam (1 mentions)
Ab16148, by Abcam (1 mentions)
Ab32575, by Abcam (1 mentions)
Ab2765, by Abcam (1 mentions)
Ab235957, by Abcam (1 mentions)
Ab103203, by Abcam (1 mentions)
Ab15580, by Abcam (1 mentions)
Cyanine3 (cy3), by Thermo Fisher Scientific (1 mentions)
Ab150079, by Abcam (1 mentions)
Ab73400, by Abcam (1 mentions)
Ab100749, by Abcam (1 mentions)
Ab2185, by Abcam (1 mentions)
Ab22048, by Abcam (1 mentions)
Ab187524, by Abcam (1 mentions)
Ab110304, by Abcam (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Ab16502, by Abcam (1 mentions)
Ab32152, by Abcam (1 mentions)
4 protocols using ab13918
1
Characterization of Heterogeneous Ovine Stromal-Derived Cells
2
Osteoclastogenesis Regulation Assay
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After culturing for 3 days, the expression of osteoclastogenesis-related genes and proteins, including NFATc1, CTSK, and RANK, was detected by qRT–PCR and immunofluorescence staining assays as mentioned earlier. The housekeeping gene GAPDH was used for normalization. The relative gene expression was calculated with the formula 2−△△CT formula and represented as a fold change relative to the control. The primer sequences are displayed in Supplementary Table 1 . To visualize osteoclastogenesis-related proteins in cells, immunocytochemistry was performed with primary antibodies at a dilution of 1:200 overnight at 4 °C. Primary antibodies, including anti-NFATc1 (Affinity, DF6446), anti-CTSK (Santa Cruz, sc-48353), and anti-RANK (Abcam, ab13918), were used in this study. Subsequently, the samples were incubated with Alexa Fluor® 647-conjugated goat anti-rabbit IgG (Abcam, ab150079) at a dilution of 1:200 in the dark for 1 h at room temperature. After staining the nuclei with DAPI, the samples were imaged by CLSM.
3
Bone Remodeling and Angiogenesis Signaling
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After collecting the cell and tissue homogenate, add to the RIPA (Thermo, 89900) lysate containing protease inhibitor, lyse on ice for 30 min, collect the supernatant, and concentrate the concentration of the collected protein using BCA (Thermo, 23225) protein quantification kit. The concentration of the solution was measured, and the protein was electrophoresed by SDS-PAGE, and OPG (ABCAm, ab73400), RANKL (ABCAm, ab100749), RANK (ABCAm, ab13918), VEGF (ABCAm, ab32152), VEGFR1 (ABCAm, ab32152), VEGFR2 (ABCAm, ab32152), HIF (ABCAm, ab2185), SIRT1 (ABCAm, ab110304), HIF-1α (ABCAm, ab187524), TLR4 (ABCAm, ab22048), NF-KB (ABCAm, ab16502) antibody were added. Incubate at 4°C overnight, wash the PVDF membrane with PBS, add horseradish peroxidase-labeled secondary antibody, incubate for 2 h at room temperature, and color the protein using ECL luminescence kit and gel imaging system. The results were analyzed by absorbance using ImageJ.
4
Histological and Immunofluorescent Analysis of Mouse Knee Joints
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The knee joint tissues of the mice were collected after upon killing and fixed in 10% buffered formalin phosphate for 48 hr. Samples were then decalcified in 10% ethylenediaminetetraacetic acid (EDTA, pH 7.4) for 2 weeks and embedded in paraffin or optimal cutting temperature (OCT) compound (Sakura Finetek). Four μm thick, sagittal-oriented sections of paraffin-embedded knee joints were processed for Tartrate-resistant acid phosphatase (TRAP) staining using a standard protocol (Sigma-Aldrich). Ten μm thick, sagittal-oriented sections of OCT-embedded knee joints were processed for immunofluorescence staining. The sections were incubated with primary antibodies against RANK (1:100, ab13918, Abcam), TLR2 (1:100, mAb12276, Cell Signaling), Maackia Amurensis Lectin (1:100, B-1265–1, Vector Laboratories), ST3GAL4 (1:100, 13546–1-AP, Proteintech), c-Fos (1:100, mAb2250, Cell Signaling), or ST3GAL1 (1:50, PA5-21721, Thermo Fisher Scientific) overnight at 4 °C. Then, the samples were incubated with secondary antibodies and DAPI (1:250, H-1200, Vector) in the dark for 1 hr at room temperature. The fluorescence images were taken by fluorescence microscopy (Olympus BX51, DP71) or confocal microscopy (Zeiss LSM 880) and analyzed by ImageJ software (National Institutes of Health, Bethesda).
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