Stim1

Fixed with 4% formaldehyde for 24 h, harvest samples were then decalcified in 10% EDTA decalcifying solution for 4 weeks and solution was refreshed each two days. Then, dehydration, embedding, and section into paraffin slices were conducted. Hematoxylin and eosin staining (H&E, Google biotechnology, China), immunohistochemistry staining (IHC, MXB biotechnologies, China) and immunofluorescence staining (IF) were carried out according to the manufacturer's protocols. For IHC and IF staining, the primary antibodies were against CD301b (eBioscience, USA), α-SMA (CST, USA), VEGF (ABclonal, China), CaN (ABclonal, China), and STIM1 (ABclonal, China). For IF staining, the anti-mouse and rabbit secondary antibodies were 594 nm red and 488 nm green fluorescent markers (Abbkine, USA). The DAPI dye (Zhongshan Biotechnology, China) was used to stain the nucleus of cells in tissue and cells. All stained sections were captured with an Olympus DP72 microscope (Olympus, Japan), and each sample was captured for 3 images of BCP bioceramics surroundings.

About 100 μL RIPA buffer containing protease inhibitor and 1% phosphatase inhibitors was used to lyse the cells and collected into the pre-cooled tube, followed by ultrasonic treatment on ice. Each sample was quantified and normalized by BCA kit (Thermo Fisher Scientific, USA). After mixed with 5X loading buffer, the protein mixture was heated for 10 min at 95 °C. Then, successive steps of separation by SDS-PAGE gelatin, transfer to nitrocellulose, soaking into 5% fat-free milk and incubation with primary and secondary antibodies (BioSharp, China) were carried out. The primary antibodies were against STIM1 (ABclonal, China), CaN (ABclonal, China), VEGF (ABclonal, China), NFATc1 (ABclonal, China) and GAPDH (ABclonal, China). At last, the results were visualized by WesternBright ECL HRP Substrate Kit (Advansta, U.S.A).