Neutral buffered 10 formalin

Porcine foetuses were obtained from artificially inseminated landrace sows (Sus scrofa). Pregnant sows were anaesthetised by inhalation of 35–70% CO₂ for 1 min and sacrificed by exsanguination. The uteri were recovered and the foetuses from 35/40, 60, 80, 100 and 115 days post-conception were quickly removed, dissected and flash-frozen in liquid nitrogen. Tissue for immunohistochemical analysis was immersed in neutral-buffered 10% formalin (Sigma-Aldrich).
Murine foetuses were obtained from timed mating of mice acquired from Taconic M&B, Ry, Denmark. Females were examined the following morning, and the stage of development was designated embryonic day 0.5 (E0.5). Pregnant mice were killed by cervical dislocation. Foetuses were dissected from the age of embryonic day 10.5, 11.5, 12.5, 13.5, 14.5, 15.5, 16.5, 17.5 and 18.5 (day of birth), and postnatal day 14 (P14) and P21. The anterior part of the embryo was collected from E10.5 to E16.5, and the brains were dissected from E17.5, E18.5, P14, P21 and from a 14-months-old adult mouse. Samples were immediately flash-frozen in liquid nitrogen. Tissue for immunohistochemical analysis was immersed in neutral-buffered 10% formalin (Sigma-Aldrich).

Quantification of circulating blood leukocytes was performed by flow cytometry as described (Kipari et al., 2013 (link)). Briefly, 30 µl of trunk blood was added to 30 µl of sodium citrate (3.9% w/v). Red blood cells were lysed by addition of 0.5 ml of FACS^TM lysis buffer (BD Biosciences, Oxford, UK), according to the manufacturer’s protocol. Fluorescent conjugated antibodies (100 ng each, in a total of 50 µl PBS) were added and incubated on ice for 25 min. The antibodies used were: CD45-PE (30-F11; Biolegend, UK), CD11b-FITC (M1/70; Biolegend, UK), Ly-6C-APC (AL-21; BD Biosciences, UK), Ly-6G-PB (1A8; Biolegend, UK). Cells were collected by centrifugation, resuspended in 100 µl of neutral buffered 10% formalin (Sigma-Aldrich, Dorset, UK) and analysed using a BD LSR Fortessa cell analyser (BD Bioscience, Oxford, UK) with manual compensation for each antibody. Supplementary Fig. 3 shows the gating strategy used to identify different classes of leukocytes. Data analysis was performed using FlowJo software v8.2 (TreeStar, USA). To determine the absolute numbers of cells, 5 × 10⁴ fluorescent flow-check fluorospheres (Beckman and Coulter, UK) were added to each sample prior to analysis. Forward and side scatter were used to distinguish cell and fluorospheres populations and cell numbers were calculated from the ratio of cells to fluorospheres in each sample.

To confirm induction of mineralisation, DPCs were stained with 2% (w/v) Alizarin Red S (Millipore, Cork, Ireland) on days 11 and 14 of culture. Briefly, the medium was aspirated, and the cells were washed with PBS. The cells were then fixed with 1 mL neutral buffered 10% formalin (Sigma-Aldrich, Arklow, Ireland) and washed three times for 5 min each with ultrapure water before being incubated with 1 mL Alizarin Red S solution (2% w/v) for 20 min. The cells were then washed three times with ultrapure, autoclaved water for 5 min each. Images of the wells were taken and stored as JPEG files, prior to solubilisation of the stain and quantification by spectrophotometric analysis at 405 nm (Tecan Genios Spectrophotometer, Unitech, Dublin, Ireland) [52 (link)]. Mineral production per cell was subsequently calculated by dividing the concentration of Alizarin Red S in each group by the corresponding number of cells counted in parallel DPC cultures using a haemocytometer and Trypan Blue staining, as described above. Three biological replicates were analysed in triplicate.