2.2-Histologic Investigation
The removed lung tissues were divided in 2 for histopathologic investigation. Some was fixed in 10% neutral formalin solution. Standard histologic techniques were applied. Later they were submerged in paraffin and sectioned to 5 µm thickness. Lung was stained with CAT, SOD, Gpx and MYP staining and investigated with a light microscope. The section allocated for electron microscope investigation was fixated in 2.5% glutaraldehyde for 24 hours at 4 ℃. Later, secondary fixation was performed in 1% osmium tetroxide at room temperature in a rotator. Tissues were washed 3 times in buffer solution, then passed through an ethyl alcohol series at 4 ℃ twice. Propylene oxide was used for transparency. Tissues were submerged in araldite the next day. The obtained blocks were sliced with an ultramicrotome and prepared for investigation. Investigation with transmission electron microscopy (TEM) assessed histopathologic changes occurring in lung tissue and especially, changes in neutrophil leukocytes.
All histologic assessments were performed by two histologists blinded to the groups.
Immunohistochemical staining in the groups was determined according to intensity (i) as (0): no staining, (1): weak, (2): moderate, (3): strong, and (4) severe staining.
Immune staining histologic scoring system (histologic score: H-score) was calculated with the following equation: H-SCORE: Ʃ Pi (i+1). 1. The H-SCOR was separately obtained for lung tissue from the multiplication of staining intensity of stained cells with percentages (Mc Carty., Miller et al., 1985).