As previously described, immunohistochemical staining was performed.21 , 22 In short, for antigen retrieval, bone sections were performed for 15 minutes by digestion with 0.05% trypsin. After that, the bone sections were incubated with primary antibody which against osteocalcin (Takara) at 4°C overnight. Later, we performed counterstaining with haematoxylin (Sigma‐Aldrich) to detect the immunoactivity. HRP‐streptavidin detection system (Dako) was made use of. As negative controls, polyclonal rabbit IgG (R&D Systems Inc) was used to incubated with sections.
Polyclonal rabbit igg
Manufactured by R&D Systems
Sourced in France
Polyclonal rabbit IgG is a laboratory reagent consisting of a mixture of immunoglobulin G (IgG) antibodies purified from the serum of immunized rabbits. It is commonly used as a control or reference standard in various immunoassays and research applications.
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5 protocols using polyclonal rabbit igg
1
Osteocalcin Immunohistochemistry in Bone Sections
2
Quantifying Osteocalcin in Mouse Femur
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Freshly, femora were dissected from mice, fixed overnight at 4℃ with 4% paraformaldehyde, decalcified in 10% EDTA (pH 7.4) for 21 days and then embedded in paraffin. Bone sections (4 μm thick, longitudinally oriented) were incubated with primary antibody against osteocalcin (Takara, M173) overnight at 4°C. Subsequently, an HRP‐streptavidin detection system (Dako) was used to detect the immunoactivity, followed by counterstaining with haematoxylin (Sigma‐Aldrich). Sections incubated with polyclonal rabbit IgG (R&D Systems) served as negative controls. Four randomly selected visual fields in the distal metaphysis of the femur were measured to test the number per millimetre of adjacent bone surface (Nmm‐1) in trabecular bone.
3
Modulation of M2-Macrophage Phenotype
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Anti‐IL‐10 (10 µg mL−1, clone JES3‐12G8, rat IgG2a, AbD Serotec, Marnes‐la‐Coquette, France), anti‐IL‐10Rα (10 µg mL−1, clone 3c.5.2b, mouse IgG1, Schering‐Plough) and anti‐PD‐L1 (20 µg mL−1, clone 29E.2A3, mouse IgG2b, Biolegend, Saint‐Cyr‐L’école, France) blocking antibodies and their respective controls were used in the suppression assay and to assess the role of IL‐10 in the modulation of surface markers on in vitro generated M2‐MΦ. Anti‐M‐CSF (20 µg mL−1, polyclonal rabbit IgG, Genzyme, Lyon, France) and anti‐TGF‐β (10 µg mL−1, pan‐specific antibody, polyclonal rabbit IgG, R&D Systems) antibodies and clinical anti‐VEGF antibody (1 µg mL−1, human IgG1, Avastin®, Roche, Bale, Switzerland) were used in SNDil‐MΦ cultures.
4
Histochemical Analysis of Murine Femur
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For histochemistry, femurs were dissected and fixed overnight with 10% paraformaldehyde at 4°C. Samples were then decalcified at 4°C using 10% EDTA (pH 7.4) for 21 days and then embedded in paraffin. Paraffin sections with the thickness of 6-mm were prepared and stained with hematoxylin and eosin for tissue histology. For immunohistochemical staining, bone sections were processed for antigen retrieval by digestion with 0.05% trypsin at 37°C for 15 minutes, and then incubated with primary antibody against Osteocalcin (Takara, M173) overnight at 4°C. The HRP-streptavidin detection system (Dako) was used to detect the immunoactivity, followed by counterstaining with hematoxylin (Sigma). Sections incubated with polyclonal rabbit IgG (R&D Systems) served as negative controls. The area used for analysis was a 1 mm 2 area within the metaphyseal secondary spongiosa.
5
Osteocalcin Immunostaining of Bone Sections
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Immunohistochemical staining was conducted, as previously described [41 (link), 42 (link)]. Bone segments were treated for antigen recovery through assimilation with 0.05% trypsin at 37°C for 15 minutes, and were then probed using a primary antibody against osteocalcin (Takara) overnight at 4°C. Consequently, an HRP-streptavidin recognition system (Dako) was utilized to distinguish immunoactivity, followed by counterstaining with hematoxylin (Sigma). The segments probed with polyclonal rabbit IgG (R&D Systems Inc.) acted as negative controls.
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