All the animals were anesthetized by intraperitoneally injecting a mixture (1 mL/kg) of Zoletil (Virbac) and Rompun (Bayer) (4:1 v/v) and perfused transcardially with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate-buffer (PB, pH 7.4). The mandible tissue inducing the periodontitis was excised and immersed in 10% paraformaldehyde in 0.1 M phosphate-buffer (PB, pH 7.4).
The mandible tissue samples from the rats were obtained by crossly trimming around the first molar teeth region, including gingival and mandibular tissues. The mandibular tissues were treated with a decalcifying solution including 24.4% formic acid, and 0.5 N sodium hydroxide for 48 h. The resulting decalcified tissues were fixed in 10% neutral buffered formalin for one day and then made into paraffin blocks using the automated tissue processor of Shandon Citadel 2000 (Thermo Scientific, Waltham, MA, USA) and the embedding center of Shandon Histostar (Thermo Scientific). From each paraffin block, five μm-thick sections were made using an automated microtome (RM2255, Leica Biosystems, Nussloch, Germany). These sections were stained with hematoxylin and eosin (H & E) according to previously established methods [41 (link),42 (link)] with our modifications and examined using the light microscope of Model Eclipse 80i (Nikon, Tokyo, Japan).
The mandible tissue samples from the rats were obtained by crossly trimming around the first molar teeth region, including gingival and mandibular tissues. The mandibular tissues were treated with a decalcifying solution including 24.4% formic acid, and 0.5 N sodium hydroxide for 48 h. The resulting decalcified tissues were fixed in 10% neutral buffered formalin for one day and then made into paraffin blocks using the automated tissue processor of Shandon Citadel 2000 (Thermo Scientific, Waltham, MA, USA) and the embedding center of Shandon Histostar (Thermo Scientific). From each paraffin block, five μm-thick sections were made using an automated microtome (RM2255, Leica Biosystems, Nussloch, Germany). These sections were stained with hematoxylin and eosin (H & E) according to previously established methods [41 (link),42 (link)] with our modifications and examined using the light microscope of Model Eclipse 80i (Nikon, Tokyo, Japan).
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