Mca497ga

Samples were fixed in 4% paraformaldehyde (PFA) overnight prior to standard processing for paraffin embedding. Serial paraffin sections were cut at 5 μm thickness. Haematoxylin and eosin, Periodic acid-Schiff, picrosirius red and Masson’s trichrome staining were performed on sections using an automated staining system (Tissue-Tek). For immunofluorescence staining, slides were dewaxed using an automated protocol and antigen retrieval was performed using citrate buffer (pH 6.0). Primary antibodies against cytokeratin 5 (CK5; Abcam; ab17130), collagen IV (COL-94; Abcam; ab6311) and laminin (Sigma; L9393) were used. Species-appropriate secondary antibodies conjugated to AlexaFluor dyes (Molecular Probes) were then used. Images were acquired using a Zeiss LSM700 confocal microscope. For immunohistochemistry, slides were dewaxed and antigen retrieved as for immunofluorescence. Primary antibodies against CD45 (Abcam; ab10558), endomucin (V.7C7.1; Abcam; ab106100) and F4/80 (Serotec; MCA497GA) were used. Sections were then incubated with species-appropriate biotinylated F(ab’)2 for 30 min, developed using ABC reagent and superDAB (Dako) and finally counterstained with haematoxylin.

After MRI experiments, animals were euthanized using an overdose of pentobarbital. Tumors were harvested and cut into 1 mm-thick slices, which were formalin-fixed and paraffin-embedded. Perls' Prussian blue staining and F4/80 immunostaining were performed on adjacent 5 μm sections to detect iron and macrophages, respectively. For macrophage detection, after manual deparaffinization, endogenous peroxidases were inhibited by a 20 min-treatment with 3% H₂O₂ in methanol. Sections were then subjected to antigen retrieval in 10 mM citrate buffer pH 5.7. Aspecific antigen binding sites were blocked with a TBS solution containing 5% BSA and 0.05% Triton. Endogenous biotin was blocked with an avidin/biotin blocking kit (Vector SP2001). Slices were stained with anti-F4/80 primary antibodies (AbDSerotec MCA497GA) overnight at 4°C at a 1/500 dilution, followed by incubation with biotinylated goat anti-rat secondary antibodies (Vector BA4001) at a 1/50 dilution for 60 min at room temperature. This reaction was visualized using Streptavidin-HRP (BD Biosciences 554066) and DAB (Dako K3468). After counterstaining with hematoxylin (Dako S3301), slices were dehydrated and coverslipped. Slides were finally digitalized at a 20× magnification with a SCN400 slide scanner (Leica, Wetzlar, Germany) and visualized on the Digital Image Hub (Leica Biosystems, Dublin, Ireland).

The aorta and PVAT were fixed with 4% paraformaldehyde and embedded in paraffin and prepared as slides (4-µm-thick sections). PVAT sections were stained with hematoxylin and eosin (HE). For the measurement of adipocyte cell size, > 250 cells were counted per each section using an image analysis software (NIH Image J software). Macrophages in the PVAT were immunohistochemically detected using a rat monoclonal F4/80 antibody (MCA497GA; Abd Serotec, Kidlington, UK). Neointima formation in cuff-injured femoral arteries were stained as immunohistochemically detected using a α-smooth muscle actin (α-SMA) antibody (ab5694, Abcam, Cambridge, UK). Elastica-van Gieson and Masson trichrome staining were also performed in femoral arteries. Binding of primary antibody was visualized using DAB + chromogen (Dako, Glostrup, Denmark). PVAT was stained using Sirius red for the assessment of fibrosis; positive areas were measured using NIH Image J software. TUNEL staining was performed using the ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instruction. TUNEL-positive cells were counted in the whole area of the section. All microscopic images were acquired using the Keyence BZ-9000 microscope.

Liver samples were fixed with neutral-buffered formalin, embedded in paraffin and cut into 4 μm thick sections that were stained with Hematoxylin and eosin and Sirius red^{22 (link)}. F4/80-positive macrophages were detected immunohistochemically using a rat monoclonal anti-mouse F4/80 antibody (MCA497GA, Serotec, UK). Apoptotic cells were detected by TUNEL assay using an Apop-Tag Plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, Billerica, MA, USA), and anti-Caspase-3 antibody (ab13847, Abcam, Cambridge, UK). Sirius red-positive area was measured using the software WinROOF (Mitani, Chiba, Japan). TUNEL-positive cells and hCLS were counted in the whole area of each section and expressed as the mean number/mm². According to the NASH clinical research network scoring system, each score for steatosis, inflammation, and hepatocyte ballooning was evaluated by two investigators^{49 (link)}. Briefly, the degree of steatosis, inflammation, and hepatocyte ballooning was scored using hematoxylin & eosin-stained liver sections. Each variable was graded from zero to three, and the sum of the scores was considered as NAFLD activity score. Fibrosis was staged from zero to three using Sirius red-stained sections.

All histological analyses were performed on fixed paraffin-embedded LV sections (5 μm). Cardiac interstitial fibrosis was assessed by picrosirius red staining (0.1 % w/v), excluding coronary vessels and perivascular regions. Data were quantified by digital image analysis (NIS-Elements, Nikon) with the observer blinded to sample identity. Immunocytochemistry for CD45 and F4/80 was performed using rat polyclonal (553076, 1:200; BD Bioscience) and rat monoclonal (MCA497GA, 1:200; AbD Serotec) antibodies, respectively, followed by secondary rabbit anti-rat IgG (P0450, 1:100; Dako) staining, using diaminobenzidine as the chromogen and nuclear counterstaining with haematoxylin.