Fixed hemi-mandible samples used for micro-CT analysis were decalcified using 10% formic acid and sodium citrate, dehydrated in ethanol and embedded in paraffin. Serial sections of 6μm thickness were cut; every 5th sample was mounted and stained with hematoxylin and eosin (H&E). Sections containing the region of interest (patent root canal with localized periapical lesion) were selected, mounted and stained with tartrate-resistant acidic phosphatase (TRAP). Methyl green staining was employed as counter staining for the TRAP staining. Adjacent sections were used for immunohistochemical analysis. Identification of neutrophils, B-cells and macrophages were performed by help of the antibodies Ly-6B.2 (dilution 1: 4000; AbD-Serotec, Oxford, UK ), CD20 (1:200, Santa Cruz Biotechnology, Inc, USA), F4/80 (dilution 1:50, Santa Cruz Biotechnology, Inc, Dallas, TX), ab3523 (anti-iNOS, dilution 1:400, Abcam, Cambridge, MA), and N20 (anti-ArgI, dilution 1:50, Santa Cruz Biotechnology, Inc) respectively. Primary antibodies were detected either with biotinylated goat anti-rat antibody or biotinylated rabbit anti-goat using the Vector ELITE ABC kits (Vector Laboratories, Burlingame, CA).
Omissions of the primary antibodies were used as negative controls and the sections showed no positive immune cell staining. Cell enumeration was carried out in grid by light microscopy at 400x magnification.
Omissions of the primary antibodies were used as negative controls and the sections showed no positive immune cell staining. Cell enumeration was carried out in grid by light microscopy at 400x magnification.
