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Identification of mature osteoclasts (mOC) on bone slices and quantification of resorbing activity 21 days after onset of the differentiation process. A <t>triple</t> <t>staining</t> for OC markers was applied <t>(TRAP,</t> red; F-actin, green; nuclei: blue; (A) . A cell was classified as mOC when it TRAP-positive and had ≥ 3 nuclei (white arrowheads; all mOC showed an actin ring, (B) , precursor (preOC, round with one or two nuclei, yellow arrowheads, (C) , proOC (spindle-shaped with a single nucleus, red arrowhead, (D) . After microscopy, cells were removed, and resorption pits were visualized by Toluidine blue staining (E) . Semi-quantitative image analysis was performed using ImageJ and masks to measure the resorbed areas per field of view (F) . Scale bar: 50 µm (A-D) , 100 µm (E, F) .
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Identification of mature osteoclasts (mOC) on bone slices and quantification of resorbing activity 21 days after onset of the differentiation process. A triple staining for OC markers was applied (TRAP, red; F-actin, green; nuclei: blue; (A) . A cell was classified as mOC when it TRAP-positive and had ≥ 3 nuclei (white arrowheads; all mOC showed an actin ring, (B) , precursor (preOC, round with one or two nuclei, yellow arrowheads, (C) , proOC (spindle-shaped with a single nucleus, red arrowhead, (D) . After microscopy, cells were removed, and resorption pits were visualized by Toluidine blue staining (E) . Semi-quantitative image analysis was performed using ImageJ and masks to measure the resorbed areas per field of view (F) . Scale bar: 50 µm (A-D) , 100 µm (E, F) .

Journal: Frontiers in Immunology

Article Title: Modulation of Differentiation and Bone Resorbing Activity of Human (Pre-) Osteoclasts After X-Ray Exposure

doi: 10.3389/fimmu.2022.817281

Figure Lengend Snippet: Identification of mature osteoclasts (mOC) on bone slices and quantification of resorbing activity 21 days after onset of the differentiation process. A triple staining for OC markers was applied (TRAP, red; F-actin, green; nuclei: blue; (A) . A cell was classified as mOC when it TRAP-positive and had ≥ 3 nuclei (white arrowheads; all mOC showed an actin ring, (B) , precursor (preOC, round with one or two nuclei, yellow arrowheads, (C) , proOC (spindle-shaped with a single nucleus, red arrowhead, (D) . After microscopy, cells were removed, and resorption pits were visualized by Toluidine blue staining (E) . Semi-quantitative image analysis was performed using ImageJ and masks to measure the resorbed areas per field of view (F) . Scale bar: 50 µm (A-D) , 100 µm (E, F) .

Article Snippet: The staining solution consists of TRAP buffer (0.1 M acetate buffer: 35.2 ml 0.2 M Sodium Acetate solution, 14.8 ml 0.2 M acetic acid, 50 ml; Millipore, Burlington, USA) mixed with 0.3 M Sodium Tartrate (pH 5.5), 10 mg/ml Naphthol AS-MX phosphate, Triton X-100, 0.3 mg/ml Fast Red Violet LB Salt (all from Sigma Aldrich Chemie GmbH).

Techniques: Activity Assay, Staining, Microscopy