Ab62461

Immunohistochemical staining was performed mainly in the humidity chamber as described in a previous study (12 (link)). Briefly, each section was pretreated with 3% hydrogen peroxide in ice-cold methanol for 20 min in order to block endogenous peroxidase activity. Next, slide-loaded sections were bathed in 0.01 M sodium citrate buffer (pH 6.0) at 95–100°C in a microwave oven for antigen retrieval. After blocking with normal goat serum for 20 min at room temperature, slides were incubated with primary anti-TRPC6 antibody (1:100; ab62461; Abcam) overnight at 4°C. Slides were subsequently incubated with the secondary antibodies, goat anti-rabbit IgG (1:200; ab205718, Abcam) and stained with 3,3′-diaminobenzidine (Boster Systems, Inc., Pleasanton, CA, USA) and hematoxylin according to the manufacturer's instructions. Finally, the slides were observed using a light microscope.

Total protein was extracted from the HDPCs or pulp tissue samples using RIPA medium containing phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology, Haimen, China). Protein concentrations were determined using a BCA Protein Assay kit (P0012; Beyotime Institute of Biotechnology). Equivalent amounts of diluted protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. After blocking for 1 h at room temperature in 5% skim milk solution, the membranes were subsequently incubated with anti-TRPC6 antibodies (1:500; ab62461; Abcam, Cambridge, UK), anti-dentin sialophosphoprotein (anti-DSPP) antibodies (1:500; sc73632; Santa Cruz Biotechnologies, Inc., Dallas, TX, USA), anti-dentin matrix protein-1 (anti-DMP-1) antibodies (1:500; sc73633; Santa Cruz Biotechnology, Inc.) overnight at 4°C. Secondary antibody anti-mouse IgG/anti-rabbit IgG, HRP-linked antibody (7076S/7074S; Cell Signaling Technology, Inc., Danvers, MA, USA) was then added at a dilution of 1:5,000 for 1 h at room temperature after the membranes were washed with Tris-buffered saline with Tween-20. Relative band intensities were detected by densitometry using Quantity One 1-D analysis software (version 4.6.2; Bio-Rad Laboratories, Inc., Hercules, CA, USA).