Slides were fixed in Hematoxylin (Sigma-Aldrich, MO, USA) for 5 min at room temperature, and then rinsed in running water for 2 minutes; after decolorizing in acid alcohol for 2 seconds, rinsed again in sodium bicarbonate for 5 dips; rinsed out container with Dehydrant after 95% iso for 30 seconds, and then fixed in Eosin (Sigma-Aldrich, MO, USA) for 2 minutes, and then washed in dehydrant 100% (Richard-Allan Scientific, MI, USA) and Xylnene (VWR, PA, USA) for 3 times.