3 3 diaminobenzidine solution

Cell counting and MTT assays as described in Noy et al.^{10 (link)} Cells were incubated with MTT for 2 h, solubilized with dimethyl sulfoxide and optical density was measured at 540 nm. For BrdU staining cells (5 × 10⁵) adhered to coverslips were incubated with 10 μm BrdU (Sigma) for 6 h and then fixed with 4% w/v formaldehyde. Endogenous peroxidase activity was blocked with 3% v/v H₂O₂ (Sigma) and then DNA denatured using 2 m HCl (Sigma). Cells were incubated with murine anti-BrdU antibody (Sigma) (1:500 in 1% w/v bovine serum albumin+10% v/v horse serum) overnight, followed by biotinylated horse anti-mouse IgG (Vector Laboratories) for 30 min and finally with Extravadin-peroxidase (Sigma) for 30 min. Cells were stained with 3,3′-diaminobenzidine solution (Sigma) and then counted.

To evaluate the expression of MMP-2 and MMP-9 proteins in the excised lung tissues, immunohistochemistry in paraffin-embedded sections was performed. In brief, the sections (5 µm) were deparaffinized and antigen retrieval was performed in citrate buffer at 98°C for 20 min, followed by incubation in 0.3% methanol/hydrogen peroxide for 15 min to quench endogenous peroxidase. Non-specific proteins were blocked with 2.5% normal horse serum for 20 min. The sections were then incubated with primary antibodies against MMP-2 and MMP-9 overnight at 4°C, followed by incubation with horseradish peroxidase-labeled secondary antibody for 30 min and avidin–biotin–peroxidase complex (Vector Laboratories Ltd., Peterborough, U.K.) for 1 h. Color was then developed by incubation with 3,3-diaminobenzidine solution (Sigma–Aldrich, U.S.A.), followed by counterstaining with Hematoxylin. The specific staining of MMP-2 and MMP-9 in the sections was observed using a microscope with a digital camera.

Xenograft tumors were fixed and sectioned. The sections were deparaffinized, probed with anti-Ki-67 antibody (Sigma-Aldrich) in a humidified chamber, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. Signals were developed using 3,3′-diaminobenzidine solution (Sigma-Aldrich). The sections were counterstained with hematoxylin.

Adipose tissues were fixed overnight in buffered formaldehyde (10%) and embedded in paraffin and were prepared at the thickness of 5 mM. The sections were deparaffinized and dehydrated with three changes of xylene for 15 min each and two changes of 100% alcohol for 5 min each. Sections were stained with haematoxylin (Sigma-Aldrich) for 1 min and differentiated in 1% acid alcohol for 10 s before counter-stained in eosin–phloxine solution (Sigma-Aldrich) for 5–6 s. Sections were then cleared in three changes of xylene for 5 min each and mounted with histofluid mounting medium (Marienfeld-Superior, Germany). Cell size quantification was performed using software by calculating pixels of adipocytes on HE staining. For immunohistochemistry, sections were sequentially incubated with primary antibody UCP-1 (Abcam, 10983, rabbit polyclonal, 0.5 ug ml⁻¹) overnight and anti-rabbit secondary antibody (Cell Signaling Technology, 7074 s, 0.1 ug ml⁻¹) for 1 h at room temperature, followed by development with 3,3-diaminobenzidine solution (Sigma-Aldrich). The nuclei were counter-stained with haematoxylin. Two independent investigators blinded to sample identity performed the staining and analyzed the adipose tissue sections respectively.

For antigen retrieval, slides were boiled in sodium citrate buffer (pH 6.0) using a microwave oven for 5 min, cooled in DW, and washed with PBS. Next, slides were treated with 3% H₂O₂ in PBS for 10 min at room temperature, followed by three PBS washes. Slides were then incubated with 0.1% Triton X-100 in PBS for 5 min at room temperature washed with PBS, and incubated with 0.01% normal serum solution for 1 h at room temperature to block nonspecific binding. Blocked slides were further incubated overnight at 4 °C with primary antibodies (Table S3) and for an additional 1 h at room temperature. Washed slides were incubated with biotinylated secondary antibodies (Vector Laboratories) for 1 h at room temperature, washed with PBS, and incubated with ABC reagent (Vector Laboratories) according to the manufacturer’s instructions. After washing, slides were developed with 3,3′-diaminobenzidine solution (Sigma-Aldrich) for 15 min to obtain a brown color. For counter-staining, slides were incubated with hematoxylin (KPNT, Cheongju, Republic of Korea) for 30 s, washed with DW, and mounted using DPX mounting solution (Sigma-Aldrich). Specimens were imaged using a slide scanner (Motic Scan Infinity 100; Motic, Beijing, China), and ImageJ software was used to measure the intensity and count positive signals [96 (link)].

Paraffin-embedded adipose tissues were prepared at the thickness of 5 μM. Deparaffinized and dehydrated sections were stained with haematoxylin and eosin (Sigma-Aldrich) as previously described29 (link). For immunocytochemistry, sections were sequentially incubated with primary antibody UCP-1 (5 μg ml⁻¹, rabbit polyclonal; Abcam, UK) overnight and anti-rabbit secondary antibody (4 μg ml⁻¹; Cell Signaling Technology) for 1 h at 23 °C, followed by development with 3, 3′ diaminobenzidine solution (Sigma-Aldrich). The nuclei were counter-stained with haematoxylin. The intensities of positively stained cells were quantified in each of five randomly selected fields by the Image J software. Two independent investigators blinded to sample identity, one investigator performed the staining and another investigator analysed the adipose tissue sections.