Immunohistochemical analysis Immunohistochemical examinations were carried out on 3 mm thick sections. For anti- KISS1R immunohistochemistry, unmasking was performed with 10 mM sodium citrate buffer, pH 6.0, at 90°C for 30 min. For anti- KISS1 immunohistochemistry, antigen unma- sking was not necessary. Sections were incu- bated in 0.03% hydrogen peroxide for 10 min at room temperature, to remove endogenous per- oxidase activity, and then in blocking serum (0.04% bovine serum albumin, A2153, Sigma- Aldrich, Milan, Italy and 0.5% normal goat serum X0907, Dako Corporation, Carpinteria, CA, USA, in PBS) for 30 min at room tempera- ture. Primary anti-KISS1 antibody (rabbit poly- clonal antibody anti-metastin [1-25)/KISS-1 (68-92), Catalog No. H-048-62, Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA] was diluted 1:1500 in blocking serum. Primary anti-KISS1R antibody (rabbit polyclonal anti- body anti-AXOR12 (375-398), Catalog No. H- 048-61, Phoenix Pharmaceuticals, Inc.,) was diluted 1:100 in blocking serum. Both antibod- ies were incubated overnight at 4°C. Sections were then washed three times for 5 min in PBS. Sections were revealed with anti-rabbit serum (DAKO® EnVision + TM Peroxidase, Rabbit, Dako Corporation) for 30 min at room temperature. Finally, sections were developed in 3,3’-diaminobenzidine (DAB, Sigma- Aldrich) and counterstained with hematoxylin. Negative controls were performed by omission of primary antibody and absorption tests. Immunoreactions detected in human placenta and rat brain were used as positive controls. The percentages of type I and II positive cells were evaluated on fields of 180¥134 mm. Five sections and 3 fields per section were examined. In rat and human carotid bodies, about 40 and 25-30 type I cells were counted for each field, respectively. The mean percentages of positive cells were calculated for each case and for the entire series. Statistics Percentages obtained were compared with Mann-Whitney test. A P value of 0.05 was con- sidered significant. Results Immunohistochemistry revealed diffuse expression of kisspeptins and receptor in type I cells of both human and rat carotid bodies. Immunostained cells were distributed both in the centre and in the periphery of the lobules. Conversely, no kisspeptins or receptor immunostainings were observed in type II cells (Figure 1). Endothelial cells of some vessels Original paper also showed moderate immunostaining for both KISS1 and KISS1R. In both human and rat superior cervical ganglia positive anti-KISS1 and –KISS1R immunostainings were selectively found in ganglion cells. Satellite cells were negative or only showed weak reaction (Figure 2). Glomic and ganglion cell immunostainings were eliminated when preabsorbed antiserum was used or primary antibodies were omitted. Discussion To the best of our knowledge, this is the first study demonstrating kisspeptin and kisspeptin receptor expression in glomic type I cells through immunohistochemistry. In the carotid body, glomic type I cells release many neuro- transmitters and peptide neuromodulators that play a role in the regulation of chemoreceptor Figure 1. Anti-KISS1 immunohistochemistry in human (A,B,E,F) and rat (C,D,G,H) carotid bodies (A-D) and superior cervical ganglia (E-H), showing selective positivity of glomic type I cells (arrows) and ganglionic cells, immunostaining being largely eliminat- ed in negative controls (B,D,F,H). Note also negativity of type II cells (asterisks) in the carotid body and of satellite cells in the superior cervical ganglia. Scale bars: A-D, 37.5 mm; E-H, 75 mm. [European Journal of Histochemistry 2011; 55:e14] [page 79]