β-Galactosidase Staining Kit

Protocol tips

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	Add 2 mL of 1X Fixing Solution. Incubate at room temperature for 5 minutes. Wash cells and add Cell Staining Working Solution. Incubate the cells at 37ºC protected from light for 1 hr to overnight.

Protocol tips
Add 2 mL of 1X Fixing Solution. Incubate at room temperature for 5 minutes. Wash cells and add Cell Staining Working Solution. Incubate the cells at 37ºC protected from light for 1 hr to overnight.

Publication protocol

H&E, safranin O/fast green, and tartrate-resistant acid phosphatase (TRAP) staining were performed using standard protocols (Sigma-Aldrich). Goldner’s trichrome staining was performed on undecalcified hard tissue sections (5 μm) following Electron Microscopy Sciences protocol. Beta galactosidase staining was performed using a staining kit (Cell Biolabs, San Diego, CA, USA; AKR-100) according to the manufacturer’s manual. Immunohistologic staining was performed using a standard protocol. Specifically, we incubated sections with primary antibodies to nestin (Aves Labs, Tigard, OR, USA; 1:300, lot NES0407), osterix (Abcam, Cambridge, UK; 1:200, ab22552), osteocalcin (Takara Bio Inc., Shiga, Japan; 1:200, M137), p-Smad2/3 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; 1:100, sc 11769), CD31 (Abcam; 1:100, ab28364), MMP-13 (Abcam; 1:100, ab3208), type X collagen (Abcam; 1:100, ab58632) and beta galactosidase (Abcam; 1:500, ab9361) overnight at 4°C. For immunohistochemical staining, a horseradish peroxidase– streptavidin detection system (DAKO, Carpinteria, CA, USA) was subsequently used to detect immunoactivity, followed by counterstaining with hematoxylin (Sigma Aldrich). For immunofluorescent staining, secondary antibodies conjugated with fluorescence were added, and slides were incubated at room temperature for 1 hour in the dark. Histological images were acquired with an Olympus DP71 microscope (Olympus Corporation, Tokyo, Japan), and quantitative analysis was performed in a blinded fashion with Image pro plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

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