Peroxidase enzyme based detection system

Whole lungs were removed, fixed by Methanol-Carnoy (Methacarn), paraffin-embedded, and 5-μm sections were obtained. Lung tissue was stained with: H&E (morphology); anti-F4/80 (macrophages); anti-CD3 (lymphocytes). Detection was carried out using peroxidase enzyme-based detection system (Vector Laboratories), and photomicrographs were taken using Nikon Eclipse 80i microscope system. Neutrophils were identified based on morphology and counted. The severity of lung injury was determined as published by Li et al.15 (link), and grading was based on the degree of inflammation and extent of lung injury: grade 0, normal tissue; grade 1, <20% of the surveyed tissue is injured and mild inflammation; grade 2, 20–50% of the surveyed tissue is injured and moderate inflammation; and grade 3, >50% of the surveyed tissue is injured with severe inflammation. The mean score from all examined fields was calculated as the injury/inflammation score (IS).

Methacarn-fixed kidney sections were dehydrated in graded alcohols and embedded in paraffin blocks using standard techniques; sections were cut, dried and rehydrated before staining. Frozen kidney tissue was embedded in OCT (Optimal Cutting Temperature), and 5 μm sections were stained with: mouse IgG (1:100 dilution); sheep IgG (1:50 dilution); mouse C3 (1:100 dilution). Paraffin-embedded and Methacarn-fixed tissue was stained with: rabbit anti-STC1 (1:300 dilution); rat anti-mouse F4/80 antibody (1:50 dilution); rabbit anti-CD3 antibody (1:200 dilution); or rabbit anti-AQP1 (1:200 dilution). Detection was carried out using fluorescence or peroxidase enzyme-based detection system (Vector Laboratories), as appropriate. Control for labeling was carried out in the presence of non-immune IgG. Photomicrographs were taken using Nikon Eclipse 80i microscope system.