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.
Hrp streptavidin detection system
Manufactured by Agilent Technologies
The HRP-streptavidin detection system is a laboratory instrument used for the detection and quantification of biotinylated molecules. It is designed to work with horseradish peroxidase (HRP) conjugated to streptavidin, a protein that binds strongly to the vitamin biotin. This system can be used in various analytical and research applications involving the detection of biotinylated targets, such as proteins, nucleic acids, or other biomolecules.
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6 protocols using hrp streptavidin detection system
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
Bone Tissue Histochemistry and Immunohistochemistry
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Histochemistry and immunohistochemistry staining was conducted as previously described.34 , 35 , 36 Femurs were fixed with 4% PFA at 4°C for 24 h, decalcified in 10% EDTA (pH 7.4) at 4°C for 3 weeks, and embedded in paraffin. 4‐μm‐thick bone sections were processed for staining. We performed HE (hematoxylin‐eosin) staining using a standard protocol. For immunohistochemistry staining, 4‐μm‐thick bone sections were blocked with 5% BSA for 60 min at 25°C after antigen retrieval and stained using primary antibody to mouse osteocalcin (Takara M173) at 4°C overnight. After that, the immunoactivity was detected using an HRP‐streptavidin detection system (Dako). The nucleuses were counterstained with hematoxylin.
4
Osteocalcin Immunohistochemistry in Bone
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The bone tissue sections were digested with 0.05% trypsin at 37°C for 15 minutes for antigen retrieval, and then the primary antibody against Osteocalcin (Takara; M173) was incubated overnight at 4°C. Subsequently, the HRP-streptavidin detection system (Dako) was used to detect the immune activity, and the counterstaining was performed with hematoxylin.
5
Immunohistochemical Analysis of Femoral Bone
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Femora were dissected from mice, carefully removing the attached muscle, and fixed overnight with 10% formalin at 4°C. After washing three times with ice-cold PBS, the samples were decalcified at 4°C using 10% EDTA (pH 7.4) for 21 d and then embedded in paraffin. 4-μm-thick longitudinally oriented bone sections of femora samples were used for staining. The sections were stained with individual primary antibodies to OCN (1:100; M137; Takara Bio) at 4°C overnight. We used the HRP-streptavidin detection system (Dako) to detect immunoactivity. Then we counterstained the sections with hematoxylin (Sigma-Aldrich). Histomorphometric analysis of two-dimensional parameters of the trabecular bones was performed using OsteoMeasureXP Software (OsteoMetrics). Osteoblast number per bone perimeter parameters were used to measure the bone formation.
6
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.
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