Immunohistochemical (IHC) staining was performed after chemical dewaxing of 4 μm thick formalin-fixed paraffin-embedded (FFPE) tissue sections as described before (30 (link), 34 (link)) using antibodies for CD14 (diluted at 1:50 in PSA) (Chemicon, CA, USA), CD68 (diluted at 1:50 in PSA) (M0814) from Dako (Agilent, CA, USA), CD163 (1:500) (Abcam, Cambridge, UK), and MAC387 (1µg/ml) (Abcam, Cambridge, UK). IHC Staining was carried out by adding 100 µl of DAB+ chromogen diluted at 1:50 in substrate buffer [(EnVision+ Dual Link System-HRP (DAB+)] for 10 min. Finally, tissue specimens were washed in phosphate buffer saline (PBS), the nuclei were counterstained with hematoxylin and mounted using Permount® (Fisher Scientific, PA, USA) for microscopic examination. Negative control slides were run in parallel with each marker where primary antibody was replaced by PBS. The stained area fractions were calculated using ImageJ software (National Institutes of Health, Bethesda, MD, USA) (7 (link), 8 (link)).
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