Cultured aggregates were fixed in 4% neutral buffered formalin for 30 min and embedded in OCT media and kept frozen. The frozen embedded samples were sectioned into 5 μm and subjected to H&E staining and Alizarin red staining. Stained sections were imaged using a camera assisted microscope (Nikon Eclipse microscope, model E6000 with an Olympus camera, model DP79). Osteocalcin (OCN) was stained as a bone marker. Briefly, sections were blocked with 5% BSA in PBS. After blocking, primary antibody anti-OCN (Abcam, Ab52128) was applied to the sections. After washing with PBS, the staining signals were visualized with Donkey anti-Rabbit IgG H&L (DyLight® 488) (Abcam, Ab96891) and imaged using a Nikon Eclipse Ti inverted microscope equipped with a Nikon DS-Fi1 camera (Nikon).
Donkey anti rabbit igg h l dylight 488
Manufactured by Abcam
Sourced in Japan
Donkey anti-Rabbit IgG H&L (DyLight® 488) is a secondary antibody conjugated with the fluorescent dye DyLight® 488. It is designed to bind to rabbit primary antibodies and can be used for immunofluorescence and western blotting applications.
Automatically generated - may contain errors
2 protocols using donkey anti rabbit igg h l dylight 488
1
Osteogenic Differentiation Analysis
2
Dual Immunofluorescence Staining for LYVE-1 and PCNA
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For double staining with anti-LYVE-1 and anti-PCNA antibodies, serial sections were processed as in “LYVE-1 immunoreactivity” until antigen retrieval. After incubation with a blocking solution for 10 min and washing with TBS-T, specimens were incubated 1 h at room temperature with anti-LYVE-1 and anti-PCNA antibodies (mouse monoclonal [PC10] to PCNA, ab29, abcam) at 2 μg/mL each. Next, slides were incubated 1 h at room temperature with Donkey Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed (1:500; abcam, ab96919) or Donkey Anti-Mouse IgG H&L (AlexaFluor® 594) preadsorbed (1:200; abcam, ab150112), and mounted with the Vector®TrueView® Autofluorescence Quenching Kit (Vector).
Stained sections were visualized using a fluorescent microscope (ECLIPSE 80i, Nikon Corporation, Tokyo, Japan), and scanned at 200× magnification to select the HPF for lymphatic vessels. To identify proliferative LECs, merged images of each channel were created using ImageJ software (version 1.52p, National Institutes of Health, Bethesda, MD, USA). For statistical analysis, four merged images of double-stained regions within each section were selected.
Stained sections were visualized using a fluorescent microscope (ECLIPSE 80i, Nikon Corporation, Tokyo, Japan), and scanned at 200× magnification to select the HPF for lymphatic vessels. To identify proliferative LECs, merged images of each channel were created using ImageJ software (version 1.52p, National Institutes of Health, Bethesda, MD, USA). For statistical analysis, four merged images of double-stained regions within each section were selected.
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