Light green sf

For Gram staining, decalcified bone samples were treated with 10 % (w/v) crystal violet (Sigma Aldrich, MO, USA) followed by Lugol's solution(Sigma Aldrich). Differentiation was performed with acetone, following by staining with 10 % (v/v) Ziehl-Neelsen's carbol fuchsin (Merck Millipore, MA, USA). Picric acid was used as a counterstain (VWR). To localise Gram-positive bacteria, a modified Gram stain was used in which samples were counterstained with 0.05 % (w/v) light green SF (Sigma Aldrich) after the www.ecmjournal.org differentiation step. To identify mycobacteria, an acid-fast bacteria stain kit was used according to the manufacturer's instructions (Dako). Tartrate-resistant acid phosphatase (TRAP) activity was visualised to demonstrate the presence of osteoclasts as described before (Croes et al., 2017a) . Immunohistochemical staining was performed as previously described in detail (Croes et al., 2017b) . A mouse-anti-human calprotectin antibody (5 μg/mL, MCA874A488, clone MAC387, Bio-rad) was used to detect activated macrophages and neutrophils. For detection of T lymphocytes, a mouse-anti-human CD3 antibody (0.7 mg/mL, M7254, clone F7.2.38, Dako) was used. For each sample, the total number positively-stained cells in one cross section was counted.