Dylight 549 goat anti rabbit igg

5 μm slides of formalin fixed paraffin embedded tissue (adenocarcinoma and chronic pancreatitis) were deparaffinized with xylol and rehydrated in ethanol. Antigen retrieval was performed in 0.05% citrate buffer, followed by wash steps in PBS. Samples were then permeabilized in 0.2% Tween, blocked in 5% goat serum and co-incubated with VDR antibody (1:100, Sigma–Aldrich) and synaptophysin antibody (1:100, Biogenex, Fremont, CA, USA) or VDR antibody and CaSR antibody (1:200, Abcam). After washing, samples were incubated with Dylight 549 goat-anti-rabbit IgG (1:500, Vector Laboratories, Burlingame, CA, USA) and Alexafluor 647 goat-anti-mouse IgG (1:1000, Jackson ImmunoResearch, Suffolk, UK). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Roche, Basel, Switzerland) and mounted with Fluoromount-G (Southern Biotech, Birmingham, AL, USA).

HT29, HT29^GFP and HT29^{CYP24A1‐GFP} cells were seeded on sterile glass coverslips and treated with 100 nM 1,25‐D₃ for 20 hrs. Post treatment, cells were fixed in 3% paraformaldehyde, permeabilized using 0.2% Triton‐X (Sigma Aldrich, USA) in phosphate‐buffered saline (PBS) for 20 min and blocked with 10% goat serum (Jackson ImmunoResearch, USA) in PBS. Cells were incubated with CYP24A1 primary antibody (1:100, SC66851, Abcam, Cambridge, UK) in 5% goat serum in PBS, for 1 hr. Rabbit IgG (Abcam, Cambridge, UK) was used as negative control. Dylight 549 goat‐anti‐rabbit IgG (1:500, Vector Laboratories, UK) was used as secondary antibody. Nuclei were stained with DAPI (Roche, Vienna, Austria) for 10 min and mounted with Fluoromount‐G (Southern Biotech, USA). Images were acquired using TissueFAXS (TissueGnostics, Vienna, Austria). Determination of the localization of the CYP24A1 was achieved by performing a colocalization staining with a mitochondrial tracker, MitoTracker RedCMXRos (Invitrogen, Grand Island, NY, USA) in HT29^{CYP24A1‐GFP} cells.