Skeletal preparations were double stained with alcian blue and ARS8 (link)10 (link). Briefly, embryos or newborns were eviscerated and the skin was removed. After fixation with 95% ethanol for 3 days, embryos or newborns were stained for 3 days in Alcian blue solution. Then they were fixed and cleared with 95% ethanol for three times and each 1.5 h, followed by treatment of 2% KOH for 3–4 h. After stained with ARS solution for 3–4 h, skeletons were cleared in 1%KOH/20% glycerol. For histological analysis, bone tissues were fixed in 4% paraformaldehyde (PFA) and then embedded in paraffin. For embryonic mice, 4 μm tissue sections were used for Von Kossa staining, DIG labelled in situ hybridization (Roche) and immunohistochemical staining (Dako). For postnatal mice, bone tissues were fixed in 4% PFA and decalcified for 2 weeks prior to paraffin embedding. Tissue sections (4 μm) were used for TRAP staining according to the standard protocol.
Immunohistochemical staining
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Immunohistochemical staining is a laboratory technique used to detect and localize specific proteins or antigens within tissue samples. It involves the use of antibodies that bind to target proteins, which are then visualized using various detection methods. This technique allows researchers to study the expression and distribution of proteins in different cell types and tissues.
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3 protocols using immunohistochemical staining
1
Skeletal Double Staining Protocol
2
Histological Analysis of Cartilage Tissue
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Hematoxylin–eosin stain and immunohistochemistry were performed as previously described [26 (link)]. Tissue sections were used for safranin O staining according to the standard protocol. Tissues were fixed in 4% paraformaldehyde for 48 h and incubated in 15% DEPC-EDTA (pH 7.8) for decalcification. Then, specimens were embedded in paraffin and sectioned at 7 μm. Immunofluorescence was performed as previously described [27 (link)]. Sections were blocked in PBS with 10% horse serum and 0.1% Triton for 1 h and then stained overnight with anti-PCNA antibody (SC-56). Donkey-anti-rabbit Alexa Fluor 488 (1:1000; Molecular Probes, A21206) was used as secondary antibodies. DAPI (Sigma, D8417) was used for counterstaining. Slides were mounted with anti-fluorescence mounting medium (Dako, S3023), and images were acquired with a Leica SP5 and SP8 confocal microscope. DIG labeled in situ hybridization (Roche) and immunohistochemical staining (Dako). OA Research Society International (OARSI) histopathological scores follow the literature that has been reported [28 (link)].
3
Comprehensive Histological Analysis of Bone
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Hematoxylin and eosin stain and immunohistochemistry were performed as previously described (7 (link)). Tissue sections were used for TRAP staining according to the standard protocol. Tissues were fixed in 4% paraformaldehyde for 48 hours and incubated in 15% DEPC (diethyl pyrocarbonate)–EDTA (pH 7.8) for decalcification. Then, specimens were embedded in paraffin and sectioned at 7 μm. Immunofluorescence was performed as previously described (33 (link)). Sections were blocked in PBS with 10% horse serum and 0.1% Triton for 1 hour and then stained overnight with anti-PCNA antibody (SC-56). Donkey anti-rabbit Alexa Fluor 488 (1:1000; Molecular Probes, A21206) was used as secondary antibodies. DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich, D8417) was used for counterstaining. Slides were mounted with anti-fluorescence mounting medium (Dako, S3023), and images were acquired with a Leica SP5 and SP8 confocal microscope. For embryonic mice, 5-mm tissue sections were used for immunohistochemistry staining, DIG-labeled in situ hybridization (Roche), and immunohistochemical staining (Dako).
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