Aperio digital pathology slide scanner

We analyzed high-resolution data from several consecutive sections across macroscopic data to control for sampling bias. Thirty-six consecutive sections (each 5 µm) through a pulmonary cavity from a representative M. tuberculosis-infected mouse were immunostained, digitally scanned at high resolution (Aperio Digital Pathology Slide Scanner, Leica Biosystems, Buffalo Grove, IL) and reconstructed in three dimensions. Each section was manually reviewed for staining characteristics. To analyze the colocalization of CD11b+ and Gr-1+ with MMPs, sequential sections were spatially registered. Immunostains were first converted to luminance, and an affine registration algorithm was utilized based on mutual information (Matlab, MathWorks, Natick, MA). Images were visualized using Amira 5.2.1 (FEI, Hillsboro, OR). A minimum of 300,000 voxels (three-dimensional pixels) were counted for each channel.

The paraffin-embedded tissue sections were maintained at 60°C for 1 h and then de-paraffinized in xylene (Sigma–Aldrich; Merck KGaA). The tissue sections were rehydrated in graded ethanol from 95 to 75% and PBS with 0.05% Tween-20 (PBS-T). For antigen retrieval, tissue slides were boiled in 10 mM citric acid buffer with 0.05% Tween-20 (pH 6.0) for 3 min at 121°C using a pressure cooker. After air cooling, the tissue sections were incubated with peroxidase blocking reagent (RTU, EnVision™+Dual Link System-HRP (DAB+); K4065; Dako; Agilent Technologies, Inc.) for 5 min, and then blocked with goat serum for an additional 30 min. Subsequently, the tissue sections were incubated with anti-FASN (cat. no. 3180), NF-κB p65 (cat. no. 8242), p-AKT (cat. no. 4060) and phosphor-p44/42 MAPK (ERK1/2) (cat. no. 4370) antibodies (Cell Signaling Technology, Inc.) at 4°C overnight followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The sections were rinsed with PBS-T, developed in 3′,3′-diaminobenzidine (DAB) substrate chromogen (EnVision™+Dual Link System-HRP(DAB+); K4065; Dako; Agilent Technologies, Inc.), and then counterstained with Mayer’s Hematoxylin (ScyTek Laboratories). All sections were scanned on an Aperio Digital Pathology Slide Scanner (Leica Biosystems).