Dapi fluoromount g

HCit immunolabelling was realized in several skin samples obtained from humans after protocol of body donation to science following death according to French regulations. The skin sample from a 77-year old man used for this article is an example showing a typical labelling. The samples were cleaned of adipose tissue and embedded in a cryomatrix (Shandon, Thermo Scientific) then frozen in liquid nitrogen before storage at -80°C. From cryomatrix block, 6-µm thick sections were performed with a microtome (MICROM Cryo-Star HM 560). After rehydration and washes with PBS, sections were incubated with anti-HCit antibody (rabbit polyclonal antibody, dilution 1:40, Covalab) overnight at 4°C. After serial washes with PBS, sections were incubated with anti-rabbit IgG antibody labelled with Alexa Fluor 488 (dilution 1:100, Invitrogen) for 30 min at room temperature. The sections were counterstained with DAPI (Dapi-Fluoromount G, Clinisciences) and hematoxylin-eosin staining.

Femur explants or femurs isolated from the mice at P16 were fixed in methanol chilled at −20 °C for 5 h or 24 h, respectively. After incubation in 0.5 mol·L⁻¹ EDTA, pH 8 for 72 h or 2 weeks, femurs were placed in 30% sucrose for 24 h, transferred to OCT compound at room temperature, and frozen in isopentane at −45 °C. The 50 μm tissue sections were permeabilized with 0.3% Triton X-100 for 30 min and immunolabeled with rabbit IgG anti-Arl13b (Proteintech #17711-1-AP, IF 1:100) or mouse IgG₁ anti-γ-tubulin (Sigma-Aldrich #T6557, 1:100) primary antibodies. The primary antibodies were detected with goat anti-mouse IgG₁ coupled to Alexa Fluor 488 (Life Technologies, 1:400) and anti-rabbit IgG coupled to Alexa Fluor 647 (Life Technologies, 1:400). Tissue sections were mounted with DAPI-Fluoromount G^® (CliniSciences). Three-dimensional images of the growth plate were obtained using a spinning disc confocal microscope. Images were displayed using FIJI and the FigureJ plugin.