F4 80

The kidneys were fixed and embedded in paraffin. The blocks were sectioned and stained with periodic acid-Schiff (PAS). Tubular injury was assessed in 5 random fields (400×) per sample. The percentage of damaged area was evaluated and scored by using a semiquantitative scale: 0, 0%; 1, ≤10%; 2, 11–25%; 3, 26–45%; 4, 46–75%; and 5, 76–100% [25 (link)]. The sections were also immunostained with antibodies against neutrophil gelatinase-associated lipocalin (NGAL; Santa Cruz Biotechnology, Santa Cruz, CA, USA), F4/80 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and 4-hydroxynonenal (4-HNE; Abcam, Cambridge, MA, USA). The percentage of area stained with NGAL or 4-HNE was analyzed by examining five random fields (400×) per sample using the i-Solution DT software version 11.0 (IMT i-Solution, Coquitlam, BC, Canada). The number of F4/80-stained cells was counted in 10 random fields (400×) per sample.

Adipose, pancreas, liver and muscle tissues were harvested after an overnight fast and fixed in 4% paraformaldehyde in 0.1 M PBS (pH 7.4). Sections were stained with haematoxylin and eosin. Perigonadal adipose tissue sections were used for TUNEL (Roche Biochemicals) and immunofluorescence for perilipin (Cell Signaling) and F4/80 (Santa Cruz Biotechnology, Inc.). For cell size and TUNEL analysis, at least 100 cells were counted per mouse. Adipocyte size was measured using ImageJ software. Number of adipocytes in the perigonadal fat pad was calculated from adipocyte diameter and fat pad mass52 (link)62 (link). Macrophages were excluded from adipocyte size and number calculations by appearance in crown-like structures or positive staining for F4/80 (refs 54 (link), 59 (link)). Pancreatic sections were immunostained for insulin and scanned by a ScanScope ImageScope system at × 20 magnification. β-Cell area was quantified using Image Scope software (Aperio Technologies)20 (link).

The detailed procedures of immunohistochemical and immunofluorescence analyses have been described previously34 (link). Protein expression was detected in OCI-Ly3 xenograft tissue sections with antibodies against F4/80 (Santa Cruz Biotechnology, Santa Cruz, CA), CD206 (eBioscience, San Diego, CA), CD31 (BD Biosciences), collagen I (Abcam), and legumain (Santa Cruz Biotechnology). Specimens from DLBCL patients were fixed and immunohistochemically stained for CD163 (BD Biosciences). Images were captured by an IX51 research microscope. (Olympus)

Immunofluorescence analysis of the pancreas after smoke exposure was performed on 5-μm-thick tissue sections mounted onto glass slides that were baked overnight at 58°C. Tissues were deparaffinized with xylene, rehydrated in decreasing concentrations of ethanol and permeabilized for 30 min with methanol solution. Antigen retrieval was performed by boiling sections in 0.5% citrate buffer for 15 min, blocked with 2.5% horse serum for 1 h and incubated with murine F4/80 and Arginase1 (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies overnight at 4°C. Slides were washed with PBS and incubated with FITC-conjugated (A-629511, Invitrogen, Grand Island, NY, USA) and Alexa-Fluor-647 (A-21447, Invitrogen)-conjugated secondary antibodies for 1 h, washed three times and mounted with Vectashield containing 4′ 6′-diamidino-2-phenylindole (DAPI).

BMDMs were prepared as described above and M1/M2 macrophages, with or without treatment with VEGF-C stimulation, were harvested, washed, suspended and labeled with DiI (Beyotime, China). Then, 1 × 10⁶ cells were transferred via the tail vein to UUO mice on the day after the operation or to ADR/CTRL mice on the 5th day. UUO mice were sacrificed on the 7th and 14th days, and the operated and lateral kidneys were collected. ADR and CTRL mice were killed on the 14th and 28th days. The kidneys were embedded in OCT compound (Sakura, Japan) and fixed with acetone. For tube-like structure observation, sections were washed with ddH₂O, blow-dried and sealed with ProLong Antifade reagent containing DAPI (Life Technology, USA). For identification of transferred cells, the sections were blocked with goat serum at room temperature, incubated with antibodies against F4/80 (Santa Cruz, USA) or LYVE-1 (Angiobio, USA) at 4 °C overnight, incubated with fluorescence-labeled secondary antibodies (Jackson ImmunoResearch) and sealed with ProLong Antifade reagent containing DAPI (Life Technology, USA).

Peritonea were fixed in 4% paraformaldehyde and then embedded in paraffin and prepared in 5 µm thick sections. To evaluate the peritoneal fibrosis, Masson trichrome staining was performed according to the protocol provided by the manufacturer (Sigma-Aldrich).
Paraffin-embedded sections (5 µm thick) were deparaffinized and rehydrated. Then antigen was retrieved at 98 °C for 10 min in 10 mM citrate buffer with pH 6 and washed with phosphate-buffered saline (PBS) for 15 min, and then the sections were treated with blocking buffer containing 5% bovine serum albumin for 30 min at room temperature before the overnight incubation with primary antibodies at 4 °C. The following antibodies were used: Collagen I (Santa Cruz, sc-293182, 1:100), CD31 (Santa Cruz, sc-376764, 1:100), F4/80 (Santa Cruz, sc-377009, 1:200). After having washed with PBS three times, the secondary antibody was added and counterstaining hematoxylin was performed, and DAB positivity was analyzed in six visual fields at 200× magnification. The positive area was measured using Image J (National Institute of Health, USA).

Neutrophil accumulation in the myocardium was stained with naphtol AS-D Chloroacetate Esterase (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's protocol. Macrophages in heart tissues were examined with the specific Ab F4/80 (1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Three slides from each block were evaluated and observed with bright-field microscopy. The results are expressed as the numbers of macrophages/field (×40 magnification).