H and e

For hematoxylin and eosin (H and E) staining, epididymal adipose tissues were fixed in 10% neutral-buffered formalin (Thermo Fisher Scientific, Waltham, MA, USA) overnight and transferred to 70% ethanol for one day. Afterwards, the tissues were processed in a routine manner for paraffin sections (Tissue Tek VIP Tissue Processor; Sakura Finetek USA, Torrance, CA, USA). Paraffin-embedded sections (5 μm) were cut and stained with H and E (Sigma-Aldrich) for microscopic examination (Olympus BX60, Waltham, MA, USA) at 20× magnification. To quantitate adipocyte size, the H and E-stained sections were analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

To perform histological examinations, the left lung was fixed in formalin after BALF sampling. The lung tissue samples were embedded in paraffin blocks, and 4 μm sections were prepared. Tissues were stained in hematoxylin and eosin (H and E, Sigma-Aldrich) and periodic acid-Schiff (PAS, IMEB Inc., San Marcos, CA, USA) to evaluate airway inflammatory responses and mucus production, respectively. A quantitative analysis of inflammation and mucus production in lung tissue was performed with the use of an image analyzer (IMT i-Solution software, IMT i-Solution Inc., Vancouver, BC, Canada).
Immunohistochemistry (ICH) was performed to evaluate inducible nitric oxide synthase (iNOS) and HO-1 expression in lung tissue by using a commercial kit (Vector Laboratories, Burlingame, CA, USA). The anti-mouse iNOS antibody (diluted 1:200, Abcam, Cambridge, UK) and anti-mouse HO-1 (diluted 1:200, Abcam) were used as primary antibodies. The slides were examined under a light microscope (Leica, Wetzlar, Germany) in a completely blinded manner.

Formalin-fixed and paraffin-embedded tumor sections were stained with hematoxylin-eosin (H and E; Sigma-Aldrich) for standard histological analysis, and sections were independently evaluated by two blinded pathologists. Tissue-infiltrating T cells were determined by immunohistochemistry using a rabbit polyclonal antibody to CD3 (Abcam, Cambridge, UK; #ab5690; diluted 1:200 in 1×TBS). Sections were stained using an Ultravision LP Detection System Kit (Thermo Scientific, Waltham, MA, USA), and color development was achieved by 3,3′-diaminobenzidine tetrahydrochloride (DAB) and hematoxylin as counterstain. Slides were observed under a light microscope (Leica DM LB2, Richmond Scientific, Chorley, UK) and photographs were taken using a Leica DFC 320 camera (Richmond Scientific). The evaluation of CD3+ T cells was performed by counting the number of positive immune cells per high power field (magnification ×400).