Ci a3 1

Immunostaining of mouse liver, spleen and lung specimens was performed as previously described with slight modification [10 (link), 17 (link)]. Briefly, after fixation with 10% formaldehyde, samples were embedded in paraffin, cut into 3.5 mm sections, mounted on glass slides coated with silane, deparaffinized in xylene and rehydrated through a series of ethanol solutions. The avidin–biotin complex method was used. For antigen retrieval, the sections were autoclaved at 98°C for 45 minutes in diluted immunosaver® (1:200). Then, specimens were incubated with normal rabbit serum (Dako; diluted to 1:75), and thereafter with a monoclonal antibody for F4/80 (CI:A3-1, Novus, Littleton, CO) (1:100) for 15 minutes using intermittent microwave irradiation [18 (link), 19 (link)]. Sections were then incubated with biotin-labeled rabbit anti-mouse IgG serum (diluted to 1:300; Dako) and a streptavidin–biotin detection kit (Ultra Tech HRP kit®, Beckman Coulter, Brea CA) sequentially. Finally, sections were stained with diaminobenzidin solution.

The hearts were fixed in 10% formalin and embedded in paraffin. Embedded tissues were cut into 4-μm sections and incubated with macrophage marker F4/80 (1:50 dilution; CI-A3-1, Novus Biologicals, Centennial, CO, United States), followed by incubation with biotin-conjugated secondary antibodies and then treated with avidin peroxidase. The reaction was developed using the 3,3′-diaminobenzidine (DAB) substrate kit. Then, the sections were counterstained with hematoxylin and finally photographed under an optical microscope (Olympus, Tokyo, Japan) at 200× magnification. A semiquantitative evaluation of F4/80 was performed using a method described in the literature (Hu et al., 2013 (link)) as follows: the proportion of positive cells was divided into five grades (percentage scores): ≤10% = 0, 11–25% = 1, 26–50% = 2, 51–75% = 3, and >75% = 4. The intensity of staining was divided into four grades (intensity scores): no staining = 0, light brown = 1, brown = 2, and dark brown = 3. Staining positivity was determined by the formula as follows: Overall scores = Percentage score × Intensity score. All measurements were carried out in a double-blind manner by two independent researchers.

We microwaved lung sections in DakoCytomation Target Retrieval Solution (Dako) for 10 minutes at 100°C and restricted endogenous peroxidase activity using the method of Brown et al.30 We immunostained sections using a Histofine Simple Stain kit (Nichirei Corporation) with rabbit polyclonal anti‐CD3 (1:400; DakoCytomation), rabbit monoclonal anti‐PAX5 (1:50; D7H5X; Cell Signaling Technology), rat monoclonal anti‐F4/80 (1:50; CI‐A3‐1; Novus Biologicals), goat polyclonal anti‐TNF‐α (1:100; Santa Cruz Biotechnology), mouse monoclonal anti‐IL‐6 (1:100; Leica Biosystems Newcastle Ltd.), transforming growth factor‐β (TGF‐β, 1:100; Santa Cruz Biotechnology), anti‐BAX (1:100; Santa Cruz Biotechnology) or mouse monoclonal anti‐8‐hydroxydeoxyguanosine (8‐OHdG) antibody (5 µg/mL; JaICA; Nikken SEIL Co., Ltd.), as previously described.21 We counted the 8‐OHdG‐positive cell numbers per field of view in lungs from each group (n = 6) in a blinded manner. Further, we compared the means of the positive values between each group using an image analysis system (MacScoop version 2.5, Mitani).