Sc68895

2-month-old mouse mandibles were dissected and fixed in 10% neutral buffered formalin at 4°C for 1 day and demineralized with 14 % EDTA solution, pH 7.5 at 4°C for 3 weeks. The tissues were then dehydrated with gradient ethanol, embedded in paraffin and sectioned at 5 m as published earlier [25 (link)]. Day 5 mouse heads were also processed in a similar manner. The following primary antibodies used were against STIM1 (610954, BD Biosciences, San Jose, CA), Orai1 & GRP-78 (sc68895 & sc376768 respectively, Santa Cruz Biotechnology, Dallas, TX), Runx2 (ab76956, Abcam, Cambridge, MA), Col1a1 (ab34710, Abcam), OPN (711215, Invitrogen), DMP1 and DPP (in house generated). Immunohistochemistry was conducted by using the VECTASTAIN ABC HRP kit (Vector Laboratories, Inc., Burlingame, CA) or the Mouse on Mouse Elite Peroxidase Kit (Vector Laboratories per manufacturer’s instructions. Colorimetry-based immunodetection was performed. For fluorescent immunocytochemistry, the secondary antibodies were Alexa Fluor 594 (ab 150080, abcam), Alexa Fluor 488 (ab 150113, Abcam) followed by the manufacturer’s protocol for further processing. Images were captured using Carl Zeiss AG Axio Observer D1 light microscope. The images were analyzed with ImageJ (1.50c) with IHC Profiler plugin to quantify positive staining. Data extracted from at least 6 images per sample were used for statistical analysis.

Anti-TRPC1 mAb (1:200, sc-133076, Santa Cruz Biotechnology), anti-TRPC3 pAb (1:400, PA5–20258, Thermo Scientific), anti-TRPC4 pAb (1:400, AB5812, Chemicon), anti-TRPC5pAb (1:1000, sc-18737, Santa Cruz Biotechnology), anti-TRPC6 pAb (1:500, SAB4300572, Sigma-Aldrich), anti-Stim1 pAb (1:1000, 4916s, Cell Signaling Technology), anti-Orai1 pAb (1:200, sc-68895, Santa Cruz Biotechnology), anti-Orai2 pAb (1:200, sc-292103, Santa Cruz Biotechnology) and anti-DARPP-32 mAb (1:500; 2306s, Cell Signaling Technology) were used. Goat anti-rabbit Alexa Fluor 488 secondary antibody (1:1000, 111–545-144), goat anti-rabbit Alexa Fluor 594 secondary antibody (1:1000, 111–585-144), HRP-conjugated anti-mouse (115–035-146) and anti-rabbit secondary antibodies (111–035-144) were from Jackson ImmunoReseach.