Imagej

Total protein, Western blot, and subsequent densitometry were performed as described by us previously [5 (link)]. Briefly, total protein was extracted from L4/L5 DRG from three rats (randomised and masked; repeated on 3 independent occasions, total n = 9 rats/group) after DC injury and pooled together to ensure enough protein. Forty micrograms of total protein was resolved on 12% SDS polyacrylamide gels and blotted onto polyvinylidene fluoride (PVDF) membranes (Millipore, Watford, UK) and probed with relevant primary antibodies: goat anti-PEDF (1:500 dilution, R&D Systems) and β-actin (1:1000 dilution, Sigma; protein loading control). Membranes were then incubated with relevant HRP-labelled anti-goat and anti-mouse IgG secondary antibodies and bands were detected using the enhanced chemiluminescence kit (GE Healthcare, Buckinghamshire, UK).
For densitometry, Western blots were scanned into Adobe Photoshop (Adobe Systems Inc., San Jose, CA, USA), and the integrated density of bands was analysed using the built-in-macros for gel analysis in ImageJ (NIH, USA, ImageJ.nih.gov/ij">http://ImageJ.nih.gov/ij) by an investigator masked to the treatment conditions [5 (link), 11 (link), 19 (link)]. Means ± SEM were plotted in Microsoft Excel (Microsoft Corporation, CA, USA).

Dentist C captured intraoral photographs weekly, and followed up with them for 4 weeks. The photographer was blinded to the process. All teeth were followed up, and there were no dropouts. All intraoral images were captured using a digital camera according to the commonly adopted method [53 (link)]. We objectively evaluated the adhesion ability by previously described methods [45 (link)]. Each photograph was normalized for the color tone, white balance, contrast, size, and angle. Moreover, each red-colored region was extracted using Photoshop CS6 (Adobe, San Jose, CA, USA) by dentist D. The extracted areas were measured in triplicate by ImageJ (ImageJ.nih.gov/ij/">http://ImageJ.nih.gov/ij/, accessed on 11 November 2015) (Figure 1). The measured values were allocated by dentist A to the HC and BC groups.

Each experiment was performed at least three biological replicates in all graphs. For fluorescence images, the intensity was quantified double‐blindly and measured using Adobe Photoshop CS6 and ImageJ. The band intensity of Western blotting or co‐IP was quantified with ImageJ. All data were expressed as mean ± SEM and were compared using ANOVA followed by a Tukey test (for experimental groups ≥ 3) or an unpaired Student's t test (for experimental groups = 2). Survival data were analyzed by log‐rank tests (Gronke et al., 2010). All statistical analysis was carried out using GraphPad Prism 5 software. A p < .05 was considered statistically significant: * indicates p < .05; ** indicates p < .01; *** indicates p < .001. All images were processed in Adobe Photoshop and assembled with Adobe Illustrator.