Hind limbs were dissected and fixed in 10% formalin for 72 hours and decalcified in EDTA (20% pH7.4) solution for 72 hours. Decalcified bones were embedded in paraffin and 5μm thick sections were collected for staining. Hematoxylin and eosin (H&E) staining was performed as previously described36 (link) and the region of interest was defined beginning 200μm distal to the tibial growth plate, extending 900μm distal to the growth plate. ROIs were quantified for bone volume using Image-Pro software (Media Cybernetics). Immunohistochemistry (IHC) for human keratin (Cell Signaling, Pan-Keratin clone C11, Catalog Number 4545, 1:500), Ki67 (Thermo Scientific, SP6, Catalog Number RM-9106-S 0, 1:250), and Pimonidazole (PIMO, Hypoxyprobe-1 Omni Kit, Hypoxyprobe, Inc, Catalog Number HP3–100Kit, 1:100) was performed according to manufacturer’s instructions using citric acid antigen retrieval. TRAP staining was performed using the acid phosphatase, leukocyte (TRAP) kit (Sigma-Aldrich) per the manufacturer’s instructions using all dilutions as specified in the kit. Quantification of TRAP staining was performed in the same ROI as bone volume above using ImageJ software. Images were quantified at 40x magnification and results are reported using a conversion ratio of 810 pixels: 100 microns.
Pan keratin clone c11
Manufactured by Thermo Fisher Scientific
Pan-Keratin clone C11 is a primary antibody that recognizes a broad range of keratin proteins, making it a useful tool for the detection of epithelial cells in various applications. It can be used to identify and characterize epithelial tissues and cells.
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2 protocols using pan keratin clone c11
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Histological Analysis of Bone and Immune Cell Markers
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Histological Analysis of Bone and Immune Cell Markers
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Hind limbs were dissected and fixed in 10% formalin for 72 hours and decalcified in EDTA (20% pH7.4) solution for 72 hours. Decalcified bones were embedded in paraffin and 5μm thick sections were collected for staining. Hematoxylin and eosin (H&E) staining was performed as previously described36 (link) and the region of interest was defined beginning 200μm distal to the tibial growth plate, extending 900μm distal to the growth plate. ROIs were quantified for bone volume using Image-Pro software (Media Cybernetics). Immunohistochemistry (IHC) for human keratin (Cell Signaling, Pan-Keratin clone C11, Catalog Number 4545, 1:500), Ki67 (Thermo Scientific, SP6, Catalog Number RM-9106-S 0, 1:250), and Pimonidazole (PIMO, Hypoxyprobe-1 Omni Kit, Hypoxyprobe, Inc, Catalog Number HP3–100Kit, 1:100) was performed according to manufacturer’s instructions using citric acid antigen retrieval. TRAP staining was performed using the acid phosphatase, leukocyte (TRAP) kit (Sigma-Aldrich) per the manufacturer’s instructions using all dilutions as specified in the kit. Quantification of TRAP staining was performed in the same ROI as bone volume above using ImageJ software. Images were quantified at 40x magnification and results are reported using a conversion ratio of 810 pixels: 100 microns.
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