Axiovision le image system

C57BL/6 mice were purchased from Jackson Laboratories. Anesthetized mice were trans-tympanically inoculated with NTHi at a concentration of 5 × 10⁷ CFU per mouse. Saline was inoculated as control. For inhibition studies, mice were injected intraperitoneally (i.p) with curcumin (50 mg/kg) 1 h prior or 1 h after NTHi inoculation. For mRNA analysis, mice were sacrificed 6 h post-NTHi inoculation. Total RNA was extracted from the dissected mice middle ear. For PMN analysis, mice were sacrificed 9 h post-NTHi inoculation. Middle ear effusions from mice were harvested with 10 μl saline (x3). Following cytocentrifugation cells were stained with Diff-Quik stain kit (Siemens) according to manufacturer’s protocol. Images were recorded with light microscopy system (AxioVert 40 CFL, AxioCam MRC and AxioVision LE Image system, Carl Zeiss). All animal studies were carried out in accordance with the guidelines of, and were approved by, The Institutional Animal Care and Use Committee at Georgia State University.

C57BL/6 mice were purchased from the Jackson Laboratory. Anaesthetized mice were intraperitoneally inoculated with 20 mg/kg resveratrol 1 hour before or 3 hours post intratracheal inoculation with NTHi at a concentration of 5 × 10⁷ CFU per mouse or saline as control. Mice were sacrificed by intraperitoneal injection with 100 mg/kg sodium pentobarbital 6 hours after bacterial inoculation. Lung tissue was harvested for total RNA extraction as previously described41 (link). For histological analysis, harvested lung tissue was fixed with 10% buffered formaldehyde, embedded in paraffin and sectioned at 4-μM thickness. Sections were then stained with hematoxylin and eosin (H&E) to visualize the inflammatory response. Stained sections were visualized and images were recorded under light microscopy systems (Axiovert 40 CFL, Axiocam MRC, and Axiovision LE Image system; Carl Zeiss) as previously described42 (link). All animal studies were carried out in accordance with the policies of, and with approval from, the Institutional Animal Care and Use Committee of Georgia State University.

For histological analysis, formalin-fixed paraffin-embedded lung tissues were sectioned (4 μm) and then stained with haematoxylin and eosin to visualize inflammatory responses and pathological changes in the lung. The stained sections were then imaged and recorded under light systems (AxioVert 40 CFL, AxioCam MRC, and AxioVision LE Image system, Carl Zeiss). The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml⁻¹ for Immunohistochemistry, 10 μg ml⁻¹ for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml⁻¹) in the paraffin section of mouse lung tissue. Blocking IRAK-M peptide (ProSci, cat# 2355 P, 25 μg ml⁻¹) was used for the negative control experiments. Epithelial cells and macrophages were recognized by antibodies of anti-E-cadherin (Santa Cruz Biotechnology, 10 μg ml⁻¹) and anti-F4/80 (Santa Cruz Biotechnology, 10 μg ml⁻¹), respectively and followed by bovine anti-goat TR (Santa Cruz Biotechnology, 8 μg ml⁻¹) incubation. Inflammation score in haematoxylin and eosin staining (Grade; 0 to 3) and IRAK-M protein expression intensity score in immunostaining (Grade; 0 to 4) were validated in a blinded fashion53 (link)54 (link)55 (link)56 (link).