Trpm7 antibody

Immunohistochemical study was performed as previously described [31 (link)]. Briefly, paraffin-embedded sections of SAN tissues were deparaffinized in xylene and rehydrated in an alcohol gradient solutions. Antigen retrieval was carried out in 0.1 M sodium citrate buffer (pH 6.0) at 95–98 °C for 10 min. Then, the tissue sections were washed, blocked for endogenous peroxidase activity, and pre-incubated with goat serum. Subsequently, the sections were incubated with TRPM7 antibody [Abcam] at 4 °C overnight, followed by a 45-min incubation with biotin-labeled rabbit anti-mouse IgG secondary antibody, and the color was developed with a DAB peroxidase substrate kit (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd). The nuclei were counter-stained with hematoxylin. As negative controls, immunostaining was performed by incubating samples with PBS instead of a primary antibody. The sections were observed under light microscope Nikon 80i Microscope (Nikon, Japan).

Cells were lysed by sonication with ice-cold lysis buffer containing RIAP protein lysate and a phosphatase inhibitor. A bovine serum albumin (BSA) protein assay kit (Beyotime, Shanghai, China) was used to detect the protein concentrations. The protein sample was separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a 4.5 μM PVDF membrane (Millipore, Shanghai, China). The membrane was blocked with 5% nonfat milk PBST and then incubated with anti-TRPM7 (1 : 1500), anti-PLC (1 : 1500), anti-P-PLC (1 : 1500), anti-SMAD1 (1 : 1500), anti-P-SMAD1 (1 : 1500), anti-COL1A1 (1 : 1500), anti-ALP (1 : 1500), anti-RUNX2 (1 : 1500), and anti-GADPH (1 : 2000) antibodies at 4°C overnight. Antibodies to PLC, Phospho-PLC, SMAD1, Phospho-SMAD1, RUNX2, COL1A1, and GADPH were purchased from Cell Signaling (Shanghai, China). The TRPM7 antibody was purchased from Abcam (Shanghai, China). After incubation, the membrane was reprobed with the appropriate secondary antibodies (conjugated with horseradish peroxidase) for 1 h. The enhanced chemiluminescence detection reagent (Thermo Scientific, Shanghai, China) and the Tanon 6600 Luminescence Imaging Workstation (Tanon, China) were used to visualize the protein bands. Western blot images were semiquantitatively analyzed with ImageJ software (NIH, USA).

Sevoflurane was purchased from Wuhan Aimeijie Technology Co., Ltd.; 10% chloral hydrate was purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd. Deoxyribonucleic acid terminal transferase‐mediated dUTP nick‐end labeling (terminaldeoxynucleotidyltransferase‐mediated dUTPnick‐end labeling, TUNEL) staining The kit and enzyme‐linked immunosorbent assay (ELISA) kit were purchased from Beijing Soleibao Technology Co., Ltd.; cleaved‐caspase‐3, bax, Bcl‐2, TRPM7 antibody, β‐actin antibody, and secondary antibody were all Purchased from Abcam; fully automatic biochemical analyzer was purchased from Berkam.