The lung tissue was fixed in 10% formalin and then embedded in paraffin. Later, the tissue blocks were cut into 5-μm sections, placed onto glass slides and stained with hematoxylin and eosin (H&E), dehydrated, and mounted. Morphologic examinations in these tissues were evaluated by light microscopy in a blinded fashion. To examine the extent of lung injury, we considered its five pathological features, such as (i) presence of exudates, (ii) hyperemia/congestion, (iii) intra-alveolar hemorrhage/debris, (iv) cellular infiltration, and (v) cellular hyperplasia. The severity of each of these pathological features was evaluated by a score indicating 0 as absent/none, 1 as mild, 2 as to show moderate, and finally 3 for severe injury. Compilations of these values obtained from individual pathological features represent the lung injury score [7 (link),32 (link)].
Gr-1 is a 21- to 25-kDa myeloid differentiation protein and also known as Ly-6G/Ly-6C. This myeloid differentiation antigen is a glycosylphosphatidylinositol (GPI)-linked protein expressed on granulocytes and macrophages. In the bone marrow, expression levels of Gr-1 directly correlate with granulocyte differentiation and maturation [33 (link)]. To examine neutrophil infiltration in lungs we performed immunohistochemistry using anti-Gr-1 Ab (BioLegend, San Diego, CA, USA; Catalog No.: 108413) as described previously [7 (link)]. In brief, 10% formalin-fixed, paraffin-embedded lung tissues were dewaxed in xylene and rehydrated in a graded series of ethanol. The slides were heated in 0.92% citric acid buffer (Vector Laboratories, Burlingame, CA) at 95°C for 30 min. After cooling to room temperature, the slides were incubated with 2% H2O2 in 60% methanol and blocked in 2% normal rabbit serum/Tris-buffered saline. Anti-Gr-1 antibody (BioLegend) was then applied and incubated overnight. Vectastain ABC reagent and DAB kit (Vector Laboratories) were used to detect the immunohistochemical reaction. Slides were counterstained with 4′, 6-diamidino-2-phenylindole and examined under a phase contrast light microscope (Eclipse Ti-S; Nikon, Melville, NY, USA). Gr-1-positive staining cells were counted in 10 visual fields/section at × 200 magnification, and averaged number was calculated.
Gr-1 is a 21- to 25-kDa myeloid differentiation protein and also known as Ly-6G/Ly-6C. This myeloid differentiation antigen is a glycosylphosphatidylinositol (GPI)-linked protein expressed on granulocytes and macrophages. In the bone marrow, expression levels of Gr-1 directly correlate with granulocyte differentiation and maturation [33 (link)]. To examine neutrophil infiltration in lungs we performed immunohistochemistry using anti-Gr-1 Ab (BioLegend, San Diego, CA, USA; Catalog No.: 108413) as described previously [7 (link)]. In brief, 10% formalin-fixed, paraffin-embedded lung tissues were dewaxed in xylene and rehydrated in a graded series of ethanol. The slides were heated in 0.92% citric acid buffer (Vector Laboratories, Burlingame, CA) at 95°C for 30 min. After cooling to room temperature, the slides were incubated with 2% H2O2 in 60% methanol and blocked in 2% normal rabbit serum/Tris-buffered saline. Anti-Gr-1 antibody (BioLegend) was then applied and incubated overnight. Vectastain ABC reagent and DAB kit (Vector Laboratories) were used to detect the immunohistochemical reaction. Slides were counterstained with 4′, 6-diamidino-2-phenylindole and examined under a phase contrast light microscope (Eclipse Ti-S; Nikon, Melville, NY, USA). Gr-1-positive staining cells were counted in 10 visual fields/section at × 200 magnification, and averaged number was calculated.
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