Additional file 1: of TLR2 and TLR9 modulate enteric nervous system inflammatory responses to lipopolysaccharide

TLR2/4/9 antibodies specifically stain ENS structures in WT mouse tissues. Tissue from WT, TLR2− / − and TLR4− / − mice was stained with TLR2/4 antibodies to test their staining specificity. In micrographs from WT mice, positive neurons were distinguished by strong immunoreactivity (white arrows) when compared to negative ones (open arrows). Knockout mice tissues displayed background immunoreactivity in neurons and unspecific secondary antibody precipitates. Pictures in this Add. File were taken in a Leica TCS SP5 microscope. Scale bars: 25 μm. The unconjugated TLR9 antibody (clone 26C593.2) raised in mouse could not be used to detect this receptor in mouse whole mounts, due to secondary antibody cross-reactivity with neurons, muscle fibres and supportive cells in the submucosa (TLR9 MOM). Conversely, the biotinylated form of this antibody displayed strong staining in the LMMP (black arrows) that was not observed in TLR9− / − preparations (Biot TLR9). Antigen retrieval was achieved by boiling sections in 10 mM sodium citrate pH 6, followed by quenching of peroxidases and avidin/biotin blocking. After primary antibody incubation overnight at 4 °C, sections were washed, incubated with ABC-HRP kit and developed with DAB (both from Vector Laboratories). Haematoxylin was used to counterstain tissues. Scale bars: 20 μm. (TIF 12107 kb)