Formalin-fixed paraffin-embedded tissue sections from M. tuberculosis-infected macaques m907, m9209 m1307, m1707, m3809, m9905, m10708, m21802, m15304, and m13207 were selected for study. All animals except m10708 had active TB at the time of necropsy. Tissue sections were deparaffinized in xylene, 100% ethanol, and 95% ethanol. Tissue sections were then placed into an antigen retrieval buffer (20 mM Tris/820 μM EDTA/0.00005% Tween 20 [pH 9.0]) containing pressure cooker (Manttra, Piscataway, NJ), incubated under pressure for 7 minutes before removal from the hotplate and allowed to cool slowly over 30 minutes. Sections were incubated in blocking buffer (2.5% BSA in PBS) for 30 minutes at 37°C, prior to addition of primary antibody diluted in blocking buffer. Antibodies for immunohistochemistry were against human CD3ε (ready-to-use format, 1:2 dilution; Dako, Carpintera, CA), CD11c (clone 5D11, 1:30 dilution; Leica Microsystems, Buffalo Grove, IL), CD68 (clone KP1, 1:50 dilution; Labvision, Freemont, CA), CD163 (clone 10D6, 1:30 dilution; Labvision), calprotectin (clone MAC387, 1:100 dilution; Labvision), HAM56 (ready-to-use format, 1:2 dilution; Enzo Life Sciences, Farmingdale, NY), iNOS (rabbit polyclonal; Labvision), eNOS (rabbit polyclonal; Labvision), arginase 1 (clone 19/arginase1, 1:100 dilution; BD Bioscience), arginase 2 (rabbit polyclonal, 1:40 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), nitrotyrosine (rabbit polyclonal, 1:100 dilution; Millipore, Billerica, MA). The specificity of iNOS and eNOS antibodies were confirmed by western blotting of iNOS and eNOS (0.1 ug/lane) in conjunction with 1 ug of M.tb lysate to confirm that the antibodies were isoform-specific and not reactive with bacterial proteins (data not shown). We were unable to identify an anti-nNOS antibody that worked for immunohistochemistry in macaque tissues and was not cross-reactive with other NOS isoforms (data not shown). Tissue sections were incubated at room temperature in cocktails of primary antibodies for 1 hr. Secondary antibodies, purchased from either Jackson ImmunoResearch Laboratories (West Grove, PA) or Life Technologies, were diluted in blocking buffer and applied to tissue sections that had been washed 3-5 times with IHC wash buffer (0.2% Tween-20 in PBS) and incubated for 1 hr at room temp in the dark. HAM56 was stained with an anti-mouse μ-chain specific secondary antibody (Jackson ImmunoResearch) that was made in donkey and not crossreactive with mouse IgG antibodies. The specificity of secondary antibodies was confirmed either by isotype or no-primary controls using the same staining and imaging protocol as sections containing stained with primary antibodies. The slides were then washed 3-5 times with IHC buffer and directly-labeled conjugates applied. Antibodies for direct labeling were chosen based on their ability to work well following significant dilution when used with a secondary antibody. The unlabeled antibodies were labeled with either Alexa Fluor 488 or Alexa Fluor 647 using the Zenon direct labeling kit (Life Technologies). Tissue sections were incubated with direct conjugates for 2 hrs at room temperature or overnight at 4°C. Slides were washed 4 times with IHC wash buffer, once with PBS and then coverslips were applied using Prolong Gold mounting medium containing DAPI (Life Technologies). Slides were cured for 24 hours at room temperature before imaging. Granulomas were imaged with either an Olympus Fluoview 500 or Fluoview 1000 laser scanning confocal microscope (Olympus, Center Valley, PA) maintained by the University of Pittsburgh’s Center for Biologic Imaging and a Fluoview 1000 laser scanning confocal microscope maintained by the University of Pittsburgh’s Microbiology and Molecular Genetics Department. Individual tissue sections from animals with active TB frequently contained multiple granulomas of various sizes and type; we chose to image granulomas with features that were representative of that particular granuloma type. Three color images (red, green, far red [pseudocolored as blue]) were acquired sequentially, followed by a DAPI image (gray) showing nuclei. Images (either single sections or serial z sections acquired at 1 μm intervals) were acquired and saved as TIFF-format images. Z series images were opened with MacBiophotonics ImageJ [available at http://www.macbiophotonics.ca/downloads.htm] or FIJI [available at http://pacific.mpi-cbg.de/wiki/index.php/Downloads] and saved as maximum intensity projections. At least three fields in the macrophage-lymphocyte region were imaged at 400-600x magnification for counting cells in tissues. Images were opened in Photoshop CS4 (Adobe Systems, San Jose CA), overlaid with a grid to facilitate analysis and counted manually by examining each channel separately or in combination for positively-stained cells. The number of nuclei per image, which was used to determine the number of cells within an image, was assessed with CellProfiler v2.0 [available at http://www.cellprofiler.org/]. Granulomas were too large to be imaged by one 200x field; consequently, multiple overlapping fields were acquired and the image of the entire granuloma assembled into a single montage using Photoshop. Preliminary work indicated that counting individual cells in granulomas for phenotypic analysis by automated or manual means was not going to be feasible due to the complexity of the environment, consequently, we used a region-based approach to analyze staining (signal) intensity in lymphocyte cuff or epithelioid macrophage regions. For analysis of region-based characteristics (macrophage markers, iNOS/Arg1 expression), non-overlapping image fields (200x magnification) of granulomas containing both epithelioid macrophage and lymphocyte cuff regions were acquired as previously indicated. From these images, regions of interest were drawn around epithelioid macrophage or lymphocyte cuff regions and the mean pixel intensity of the red, green, and blue channels was determined with Photoshop’s histogram tool. The iNOS:Arg1 ratio was calculated by dividing the mean iNOS signal by the mean Arg1 signal. Pairwise comparisons were made between macrophage surface marker signal or iNOS:Arg1 signal ratio were made between the epithelioid macrophage and lymphocyte cuff regions.