Immunohistochemistry (IHC) (A-C) for TLR9 detected by a mouse monoclonal antibody. Adenocarcinoma of the lung (A). Squamous cell carcinoma of the lung (B). A549 cells (all 600 ×) (C). In situ hybridization (ISH) targeting mRNA of human TLR9 with a digoxigenin-labeled DNA-probe in a squamous cell carcinoma of the lung (600 ×) (D). Immunohistochemical staining of TLR9-expression-levels in nonmalignant (E) and malignant tissues (F) derived from the same lungs an stained by the use of tissue arrays. Results of RT-PCR targeting TLR9 in cell lines (G). M: molecular-weight marker (MW8, Roche). 1: negative control; 2: A549; 3: NCI-H727; 4: BEAS 2b; 5: Mononuclear cells from a healthy human donor. Confocal laser microscopy of A549 cells transiently transfected with a GFP-TLR9 plasmid: Cytoplasmic expression of TLR9 is observable in all cells, while successful transfection led to overexpression of TLR9 resulting in bright GFP signals completely superimposed by the TLR9 antibody signal (H). Nuclear counterstain was performed with TOTO3.