S72 Fig. 7 Distribution pattern of P2X3 immunoreactivity in peptidergic and non-peptidergic small-diameter c-fibers in dorsal root ganglion (DRG) and nodose ganglion (NG) neurons. Expression of P2X4 in activated spinal microglia following peripheral nerve injury (PNI). a DRG sections were stained with P2X3 (red, #APR-016) and substance P (SP, green) antibodies (upper panel) or with P2X3 (red) and isolectin-B4 (IB4, green) antibodies (lower panel). Pink arrows indicate P2X3-positive cells with IB4 or SP immunostaining, and white arrows indicate P2X3-positive cells without IB4 or SP binding. b Immunohistochemical staining comparing P2X4 expression in sham and injured mice using anti-P2X4 receptor antibody (#APR-002, green). P2X4 expression was barely detectable in sham animals, whereas 10 days post-PNI, it was upregulated ipsilateral to the lesion. Scale bar, 500 μm. c P2X4 expression (red) in activated microglia following peripheral nerve injury is co-localized with microglia- specific eGFP fluorescence in CX3CR1+/GFP mice 10 days post-PNI. Scale bar, 100 μm. a Adapted from reference 61 with permission of Springer. b–c Adapted from reference 63 with permission of Society for Neuroscience Purinergic Signalling (2012) 8 (Suppl 1):S61–S79 microglia in nerve injured P2X4−/− mice was not affected [63]. However, in P2X4−/− mice with spared nerve injury (neuropathic pain model), tactile allodynia was significantly reduced compared with wild-type mice, indicating a possible role for P2X4 in chronic pain [63]. Apart from their importance as a tool in proteomics studies by identifying protein expression, tissue distribu- tion, and more, antibodies can also be used as a tool for determining structural and functional interaction. Assembly and trafficking of the P2X4 and P2X7 channels were demonstrated in microglia cells prepared from cerebral cortices of newborn rats using anti-P2X4 receptor and anti- P2X7 receptor (#APR-004) antibodies [64]. Using cross- linking agents, biotinylation technique, Blue native PAGE, and specific antibodies, it was demonstrated that in primary cultures of rat microglia, P2X4 and P2X7 receptors preferentially assemble as homotrimers [64]. Analysis of P2X4 and P2X7 expression on the plasma membrane of primary microglia cells indicated that P2X4 resides pre- dominantly within intracellular compartments, while P2X7 showed a prominent expression at the plasma membrane. The activation of the microglia with lipopolysaccharide increased the stability of P2X4 expression on the plasma membrane within 3 h post-stimuli followed by a modest increase in the overall expression of the channel, as demonstrated by Western blot analysis. The use of subunit-specific antibodies enables the detection of the different P2X subunits in tissues as well as in primary cultures or cell lines. In rat cultured cortical astroglia, immunohistochemical studies demonstrated that