Naphthol as bi

The TRAP activity was performed to mark the osteoclasts^8,19. The sections were deparaffinized, hydrated, and placed in a solution of 50% ethanol/acetone for 1 minute and dried at room temperature. Next, a buffer solution containing acetic acid, dimethylformamide, Fast Red, and phosphoric acid naphthol AS-BI (Sigma-Aldrich Corporation, St. Louis, MO, USA) was pipetted over the sections, which were maintained at 37°C for 40 minutes protected from light. The counter-stain with Fast Green was performed.
The samples were examined under the Axio Imager. M1 microscope under conventional light to count the number of multinucleate TRAP-positive cells present in the resorption lacunae that were in direct contact with the alveolar bone around the periapical lesion.
There was an invagination of partially mineralized connective tissue into the root canal in the ANP specimens. In the healthy specimens, the osteoclast count region was established as described for the fluorescence microscope morphometry. All results were expressed in cell numbers.

Fixed tibiae were stored at 4°C in 70% ethanol prior to decalcification in 10% ethylenediaminetetraacetic acid (EDTA) for 7 days at 4°C. Specimens were then embedded in paraffin, sectioned (5 μm) on a microtome, stained with hematoxylin and eosin (H&E), and examined under a microscope. Tumor burden was quantified using ImageJ software and was reported as a percentage of the total free space in the marrow cavity. For osteoclast counts, bone sections were stained for tartrate-resistant acid phosphatase (TRAP) utilizing a substrate incubation step (0.2 mg/mL Naphthol AS-BI, Sigma-Aldrich) followed by a color reaction (25 mg/mL Pararosaniline dye, Sigma-Aldrich) to form a bright red stain in TRAP-positive cells. Sections were then counterstained with hematoxylin, coverslipped, and examined under a microscope and quantified using ImageJ. Osteoclasts were identified as TRAP-positive, multinucleated cells juxtaposed to bone. IHC was carried out on decalcified paraffin-embedded tibial sections by Vanderbilt's Translational Pathology Shared Resource. Positive staining of FOXP3 was quantified by ImageJ.

Slides were put into xylene for 5 min three times, shook in 100%, 90%, 80% and 70% ethanol for 2 s two times and washed in the water for 10 min. Next, the reaction solution was made by 5 mg of Naphthol AS-BI (N2250-1G, Sigma-Aldrich, St. Louis, MO, USA), 0.15 mL of N.N-Dimethyl Formamide (Guaranteed Reagent 10344 Kanto Chemical Co. Inc., Tokyo, Japan), 25 mL of D.W., 0.2 mol acetate buffer (pH = 5.4) and 0.383 g of L (+)-Tartaric Acid (M6T3632 Nacalai Tesque, Inc., Kyoto, Japan). After that, the solution was kept on rotation for 10 min, and the pH was adjusted to 5.2 by 1 mol/L-Sodium Hydroxide Solution (L8E4959 Nacalai Tesque, Inc., Kyoto, Japan). After filtering the solution with filter paper, the slides were kept in the solution and warm in hot water bath for 30 min. The TRAP-stained sections were examined using an optical microscopy for histological evaluation.