Arrows show neurons exhibiting double immunopositivity, whereas asterisks indicate neurons with p-CREB immunopositivity without expressing Nav1.7. using Nav1.7 blockers should be considered to control pain in burn injury. Key messages ? Burn injury upregulates Nav1.7 expression in main sensory neurons. ? Burn injury results in increased?activity of KX-01-191 Nav1.7-expressing main sensory neurons. ? Inhibiting Nav1.7 by protoxin II reduces spinal nociceptive processing. ? Nav1.7 KX-01-191 represents a potential target to reduce pain in burn injury. Electronic supplementary material The online version of this article (10.1007/s00109-017-1599-0) contains supplementary material, which is available to authorized users. tests were used. In all cases, differences were considered significant at test and test with Bonferroni correction for multiple comparisons. The sEPSC frequency after ProTxII application was also normalised against the pre-application control value. Data are expressed as mean??standard error of mean; refers to the number of biological repetitions. Results Antibody specificity In our pilot experiments, we tested several anti-Nav1.7 antibodies and immunolabelling procedures on sections cut from rat L4 and L5 DRGs. While all the KX-01-191 antibodies provided similar staining pattern, the antibody supplied by Millipore (Supplementary Table 1) and the procedure described above produced the highest signal-to-noise ratio, which we found suitable for quantitative analysis. In WT mice, a significant proportion of neurons appeared immunopositive in the cytoplasmic compartment as well as in the cytoplasmic membrane (Fig.?1a). In contrast, only a few immunopositive neurons were apparent in sections slice from Nav1.7-cKO mouse DRG (Fig. ?(Fig.1b).1b). The presence of a limited quantity of Nav1.7-immunopositive neurons was expected in Nav1.7-cKO mouse DRG, as neurons that do not express Nav1.8 may express Nav1.7 . No immunostaining was observed in unfavorable controls either when the primary antibody was replaced by normal serum on sections slice from WT mouse DRGs (data not shown) or when it was exhausted with the immunising peptide. Open in a separate windows Fig. 1 The anti-Nav1.7 antibody specifically identifies a sub-population of main sensory neurons. a Incubation with the anti-Nav1.7 antibody (Millipore, AB5390) results in immunostaining in a group of main sensory neurons in wild-type (WT) mice. Asterisks show immunopositive neurons. b The same anti-Nav1.7 Rabbit Polyclonal to IKK-gamma (phospho-Ser31) antibody produced only?faint?staining in very few neurons?in sections cut from your dorsal root ganglia dissected from mice lacking Nav1.7 (KO; asterisks). c The anti-Nav1.7 antibody also produces immunostaining in rat primary sensory neurons. Immunopositive cells are indicated by asterisks. d Cell size distribution of Nav1.7+ neurons in the L4 and L5 dorsal root ganglia of naive Sprague-Dawley rats. Note that most of the Nav1.7+ neurons are small- and middle-size cells. Vacant bars show size distribution of all cells whereas reddish bars indicate the size distribution of neurons exhibiting Nav1.7 immunopositivity. Though we have not tested the expression of functional Nav1.7, the specificity and selectivity of the antibody suggest the expression of such functional channels. Scale bar?=?50?m on each microphotographs (Color physique online) In rat L4CL5 DRGs, a significant proportion of neurons exhibited immunostaining (Fig. ?(Fig.1c).1c). No immunopositive neurons were visible when the primary antibody was replaced by normal serum (data not shown) or worn out with the immunising peptide (Supplementary Fig.?1a). In the positive control, an already characterised antibody (anti-TRPV1 antibody; ) showed the characteristic immunopositivity (Supplementary Fig.?1b). Nav1.7 immunopositivity was found in about 25% of the neurons in naive rat DRGs (25.69??6.03%, test). Open in a separate window Fig. 2 Burn injury induces upregulation in Nav1.7 expression. a A gel image of Western blotting using the anti-Nav1.7 KX-01-191 and anti–tubulin III antibodies with protein samples isolated from your ipsilateral (ipsi) and contralateral (contra) L4 and L5 dorsal root ganglia of a rat 180?min.