Histological and Immunofluorescence Analysis of Embryonic Development

For histology and immunofluorescence, the heads of the pups and embryos were fixed overnight in 4% PFA, dehydrated to 100% ethanol, embedded in paraffin, and sectioned into 5-μm-thick slices. Immunohistochemical and immunofluorescence staining was performed with anti-Hdac4 polyclonal antibody (ab12172, Abcam, United States, 1:250), anti-Runx2 antibody (sc-390351, Santa Cruz, United States, 1:50), anti-Ki67 rabbit monoclonal antibody (ab16667, Abcam, 1:200), anti-P21/Cdkn1a mouse monoclonal antibody (sc-6246, Santa Cruz, 1:50), goat polyclonal secondary antibody to rabbit IgG (ab150078, Abcam, 1:500), and goat polyclonal secondary antibody to mouse IgG (ab150114, Abcam, 1:500) following a previously described protocol (Zhao et al., 2020 (link)). Images were captured using an Olympus IX83 inverted microscope.

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Ha N., Sun J., Bian Q., Wu D, & Wang X. (2022). Hdac4 Regulates the Proliferation of Neural Crest-Derived Osteoblasts During Murine Craniofacial Development. Frontiers in Physiology, 13, 819619.

Publication 2022

ab12172 sc-390351 ab16667 sc-6246 ab150078 ab150114 IX83 inverted microscope

Anti antibody Cdkn1a Dehydrated ethanol Embedded paraffin Embryos Goat Heads Igg antibody Immunofluorescence Microscope Monoclonal antibody Mouse Rabbit Runx2

Corresponding Organization : Shanghai Jiao Tong University

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Fixation of heads of pups and embryos in 4% PFA overnight
Dehydration to 100% ethanol
Embedding in paraffin
Sectioning into 5-μm-thick slices

dependent variables

Hdac4 expression
Runx2 expression
Ki67 expression
P21/Cdkn1a expression

control variables

Immunohistochemical and immunofluorescence staining protocol
Imaging using Olympus IX83 inverted microscope

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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