Axioima

Histological analysis was performed as previously described (36 (link)). Briefly, samples were embedded in paraffin and sagittal sections of 5μm were cut through the extraction site, then stained with hematoxylin and eosin. Three random microphotographs were taken at 20x magnification of the region of interest (ROI) per animal (Zeiss AxioIma, Carl Zeiss Microscopy GmbH, Jena, Germany). The ROI was selected at the base of the extraction defect, using the mesial root of the third molar as a reference point, where the length of the ROI was determined by the beginning and the end of the root. The number of dead osteocytes (empty osteocyte lacunae) divided by the total number of cells were counted by a blinded investigator, and results were presented as percentage:
$\frac{\mathbf{Number}\mathbf{of}\mathbf{empty}\mathbf{lacunae}}{\mathbf{Total}\mathbf{number}\mathbf{of}\mathbf{lacunae}}\times 100$ . The degree of alveolar bone necrosis, quantified as the percentage of empty lacunae at the extraction sites, was compared between the saline and EDTA sides in the control and Zol-treated animals.

IHC was done as previously described (19 (link)). Briefly, 5 μm sections of formalin-fixed paraffin-embedded tissue were de-paraffinized and rehydrated with ethanol. Antigen retrieval was done in water bath heating at 97° with citrate buffer PH6 (Thermofisher). Immune labeling was done using overnight incubation at 4° with anti ITGAX (CD11C) 1:20 dilution (Myebiosource, San Diego, USA) and anti MHCII 1:100 dilution (abcam, Cambridge, USA). Detection of the primary antibody was done with Horeseradish peroxidase (HRP) and Diaminobenzidine (DAB) substrate (Thermofisher). Multiple random photomicrographs were taken for each lymph node in the paracortex region and gingival tissues in the lamina propria, at 40x objective lens using Zeiss microscope (Zeiss AxioIma, Carl Zeiss Microscopy GmbH, Jena, Germany) and quantification was done blindly using automated Image-J software (https://imagej.nih.gov/ij/) with a set threshold based on the negative control.