Bx60f5 microscope

Immediately following death due to allergen exposure, lungs from CC011 mice were excised and fixed in 10% neutral buffered formalin. For mice exposed to PBS for 5 weeks, lungs were excised 72 h post final exposure and fixed in 10% neutral buffered formalin. Lungs were cut in 2 mm cross sections along the main stem bronchus starting at the hilum. Sections were embedded in paraffin, cut in 5 micron sections, and stained with hematoxylin and eosin (H&E), Alcian-blue and Periodic acid-Schiff (AB-PAS), or Mason’s Trichrome. Immunohistochemistry was performed for α-smooth muscle actin (α-SMA, Abcam ab124964). Sections from the most proximal lung section were imaged on an Olympus BX60F5 microscope with CellSens Standard software (Olympus Life Sciences).

LIV used for Oil red O (ORO) staining were rapidly dissected and stored in 6% buffered formaldehyde (Histolab, Gothenburg, Sweden) for 48 hours prior to sucrose treatment for dehydration. Afterwards, the samples were embedded in OCT and subsequently sectioned followed by staining with ORO and hematoxylin to assess lipid content and morphology. ORO area was computed by an image analysis program (CellSens Dimension Desktop 1.5, Version 1.5, Olympus Optical Company, Hamburg, Germany). Representative micrographs were captured with an Olympus BX60F5 microscope with a 10X objective, connected to an Olympus DP72 camera.

Macrophages infiltrating WAT arrange around dead adipocytes, forming crown-like structures (CLS) [14 (link)], therefore we stained WAT for macrophage marker F4/80. Adipose tissue was fixed in 4% formaldehyde, embedded in paraffin and sectioned. Sections were then deparaffinized and rehydrated. Antigen retrieval was performed by incubation in citrate buffer, pH 6.0, for 15 minutes, and endogenous biotin was blocked using avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA, USA) for 15 minutes. Endogenous peroxidase activity was quenched by 30 minutes incubation in 0.6% hydrogen peroxide. Staining was performed using a primary F4/80 rat anti-mouse antibody (1:20, AbD Serotec, Raleigh, NC, USA) followed by a biotinylated rabbit anti-rat secondary antibody (1:200, Vector Laboratories). Binding of secondary antibody was visualized using an avidin biotinylated-horseradish peroxidase complex (Vector Laboratories) followed by DAB staining (Dako, Glostrup, Denmark). Sections were counterstained with Mayers hematoxylin. Images were obtained with a MIRAX Scan (Carl Zeiss, Göttingen, Germany) and analyses were done using BioPix iQ software (version 2.1.4., BioPix, Göteborg, Sweden). Representative micrographs were captured with an Olympus BX60F5 microscope with a 10X objective, connected to an Olympus DP72 camera.

Cells were fixed in absolute methanol for 10 min at -20°C, washed in PBS and treated with 2 N HCl for 1 h at 37°C. The material was then washed twice in borate buffer (100 mM boric acid, 75 mM NaCl and 25 mM sodium tetraborate, pH 8.5) and blocked with 1% BSA in PBS for 1 h. Next, the cells were incubated with mouse anti-5-methylcytosine primary antibody (Sigma^®, 1:100 diluted in 1% BSA) for 1 h at room temperature in the dark, followed by treatment with goat anti-mouse IgG conjugated to FITC (Sigma, 1:50 diluted in 1% BSA) for 1 h in the dark.
Image capture was performed using an Olympus BX60F5 microscope, QCapture and Image Pro-Plus software, and the same exposure times. ImageJ (NIH, Bethesda, USA) software was used for image analysis.

Immunostaining was performed on formalin-fixed cross-sections of the aortic root as previously described [14 (link), 17 (link)]. The following antibodies were used: Mac-2 (CL8942AP: Cedarlane Laboratories Ltd., Burlington, Ontario, CA) and biotinylated rabbit anti-rat IgG (BA-4001: Vector Laboratories, Burlingame, CA, USA). Binding was visualized by DAB kit (Vector Laboratories) and counterstained with Hematoxylin (HARRIS HTX Histolab: Histolab Procuts AB, Gothenburg, Sweden). Images were produced with an Olympus BX60F5 microscope with a 10X objective connected to an Olympus DP72 camera. Positive staining for Mac-2 was automatically traced using cellSens Dimension analysis software (Version 1.5, Olympus Optical Company, Hamburg, Germany) and normalized to lesion area. Data are presented as the average staining from two consecutive sections.