2.8 | Immunohistochemical assessments
Lung sections (5 µm) from paraffin-embedded tissues were used for immunohistochemical studies. After deparaffinization and dehydration, endogenous peroxidases were blocked with 5% H2O2, nonspecific protein-binding sites were blocked with Thermo Scientific Ultra V block (TA-060-PBQ, ThermoFisher Scientific, Pittsburg, USA), and the slices were permeabilized with 0.5% Triton X-100 (Merck, Kenilworth, USA) in Tris-buffered saline. The slices were incubated with rabbit anti-myeloperoxidase or anti-tumor necrosis factor (TNF)-α primary antibody (CAT: Abcam, ab6671, 1:50; Abcam, ab9535, 1:100, Cambridge, MA, USA) overnight at 4 °C. The subsequent steps of incubation included an enhancer reagent and horseradish-peroxidase-linked secondary antibody, both of which are components of the Polink-2 Plus HRP Detection Kit (D39, GBI Labs, Bothell, WA, USA). The staining was visualized with diaminobenzidine. The slices were mounted on microscopic slides with Eukit® (Merck, Kenilworth, NJ, USA) and digitally recorded with a microscope slide scanner (Zeiss Mirax Midi Slide Scanner, Carl Zeiss MicroImaging GmbH, Germany) operated by a CaseViewer software (3D Histech Ltd., Hungary).
Myeloperoxidase is a biomarker of neutrophil activation, and TNF-α is an important cytokine in LPS-induced lung inflammation.36,37 The levels of myeloperoxidase and TNF-α expression in lung sections were assessed with a semiquantitative approach. The percentage of positive staining cells per slide (0% to 100%) was multiplied by the dominant intensity pattern of staining (0, negative or trace; 1, weak; 2, moderate; 3, intense). The maximal score was 300. All the histological sections were examined by Aperio Color Deconvolution v9 (Leica Microsystems, Germany).