Dm 480 camera

The left lung was removed from each mouse, fixed with 10% formalin solution, embedded using Tissue-Tek® (Sakura Finetek®, Torrance, CA, USA), then sectioned using a Leica microtome 820 (Leica Microsystems, Wetzlar, Germany) at 4 μm (n = 6). The sectioned tissues were stained with hematoxylin–eosin (H&E). The degree of inflammation in lung tissues was determined by the inflammation scoring system, where 0 = no inflammation, 1 = occasional cuffing with inflammatory cells, 2 = most bronchi or vessels surrounded by a thin layer (1–5 cells) of inflammatory cells, and 3 = most bronchi or vessels surrounded by a thick layer (>5 cells) of inflammatory cells. Periodic acid–Schiff (PAS) staining of the lung tissues was performed using the PAS Stain Kit (Abcam, Cambridge, UK). To quantify mucus production, mucus secretion was evaluated by measuring the broncho-alveolar red-stained regions using the ImageJ software program (NIH Image, Bethesda, MD, USA) [37 (link)]. Congo red staining of the lung tissues was performed using the Congo Red Stain Kit (Abcam). Eosinophils were counted in an area of 20,000 μm² of lung tissues after staining with Congo red [38 (link)]. Six random fields of each stained tissue section were observed under a microscope (Leica Microsystems), and images were captured using a Leica DM 480 camera (Leica Microsystems).

MIP-1β protein was detected using 20 μg/ml rabbit anti-CCL4 polyclonal antibody (Invitrogen, Carlsbad, CA, USA, catalog number PA5-34509, lot #UL2898185) and 24 μg/ml goat anti-rabbit IgG FITC secondary antibody (Invitrogen, catalog number 65–6111, lot #UG285467) in the 4-μm paraffin section of mouse lung tissue (n = 6). The nucleus was stained using UltraCruz® Aqueous Mounting Medium with DAPI (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Six random fields of each immunofluorescence-stained tissue section were observed under a microscope (Leica Microsystems), and images were captured using a Leica DM 480 camera (Leica Microsystems). The fluorescence intensity was measured using the ImageJ software program (NIH Image).

The ear tissues were removed from each mouse, fixed with 10% formalin solution, embedded using Tissue-Tek^® (Sakura Finetek^®, Torrance, CA), and then sectioned using a Leica microtome 820 (Leica Microsystems, Wetzlar, Germany) at 4 µm (n = 3). The sectioned tissues were stained with haematoxylin–eosin (H&E). Six random fields of each stained tissue section were observed under a microscope (Leica Microsystems, Wetzlar, Germany), and images were captured using a Leica DM 480 camera (Leica Microsystems, Wetzlar, Germany) and processed using Leica Application Suite (LAS) software (Leica Microsystems, Wetzlar, Germany).