S76 activity is maintained in brain preparations originating from P2X7 KO mice. For example, two bands of 65 and 77 kDa are detected in Western blot analysis using anti-P2X7 receptor antibody in the wild-type mouse whole brain, which is maintained in brains from KO animals, while the single 77-kDa band that is detected in peritoneal macro- phages of wild-type animals is abolished by the KO [76]. In addition, P2X7 immunoreactivity which is detected on cerebellar granule cells from wild-type mice in culture is seen also in such cells from KO animals. P2X7 is expressed in the developing rat brain from embryonic day 14 and might be involved in programmed cell death during embryogenesis. Immunoreactivity of the P2X7 receptor protein in the developing brain was detected in the lateral hypothalamus at E14, the lateral olfactory tract and optic tract and in the cerebellum, midbrain, and medulla at E18 [88]. P2X7 was also detected by Western blot analysis in rat primary cortical neurons in culture, which were confirmed as such by positive staining with monoclonal antibody against the neuronal marker NeuN. The P2X7 positive signal was reduced once the cells were treated with a specific antisense oligonucleotide. Functional studies with specific pharmacology have suggested that extracellular ATP mediates neuronal apoptosis through the activation of the P2X7 receptor and its downstream signaling pathways involving JNK1, ERK, and caspases 8/9/3 [71]. P2X7 expression and role in other tissues The expression of P2X7 was confirmed in calvarial osteoblasts (skull bone cells) in Western blot analysis using anti-P2X7 receptor (extracellular) antibody, a signal which was absent in the same cells from KO mice. In addition, P2X7 was expressed in MC3T3-E1 osteoblasts and MLO- Y4 osteocytes where it is activated following mechanical load, leading to prostaglandin release from these cells and the subsequent bone formation [80]. P2X7 expression was also confirmed in synoviocytes from rheumatoid arthritis patients by Western blot analysis. Pharmacological activa- tion of P2X7 in these joint cells leads to increased IL-6 secretion, suggesting a role for P2X7 in a pro-inflammatory pathway [69]. P2X7 was also reported to be expressed in skeletal muscle cells: In mouse myotubes in culture, P2X7 immu- noreactivity was confirmed by immunocytochemistry and Western blot analyses; it was shown that the P2X7 signal is significantly reduced following specific siRNA treatment. Combining these results with functional studies, it is suggested that the activation of ionotropic P2X4, P2X5, and P2X7 and metabotropic P2Y1 and P2Y4 receptors participates in forming the calcium transients of multinu- cleated myotubes [89]. Purinergic Signalling (2012) 8 (Suppl 1):S61–S79 In pancreatic cancer and chronic pancreatitis patients, some evidence for P2X7 upregulation is observed in comparison to normal pancreas. This upregulation was statistically signifi- cant in RT-PCR experiments, but not in comparative Western blot or immunohistochemical analyses. A detailed examina- tion of immunohistochemistry experiments demonstrated that most of the P2X7 immunoreactivity arises from staining of immune system-infiltrating cells and was absent from parenchymal cells [90]. In the insulinoma cell line INS-1e, P2X7 expression was detected by Western blot analysis (which was in correlation to mRNA levels), which increased following P2X7 agonist application. However, P2X7 recep- tors are probably not involved in insulin secretion or apoptosis induction in these cells [91]. Recently, a novel P2X7 splice variant (P2X7k) with an alternative first transmembrane domain was discovered. Interestingly, this splice variant escapes gene inactivation in KO mice, and its expression in different tissues (including brain) of both wild-type and P2X7 KO mice was confirmed using specific RT-PCR primers. In addition, at the protein level, its expression was confirmed using Western blot analysis in spleen, salivary gland, and lung from wild-type mice as well as in the spleen of KO mice [73]. Ionotropic and matabotropic purinoceptors have indeed proven to be most important in various biochemical and physiological processes, as pointed out in the above examples. Importantly, the pharmacological tools that have been used to unravel these important receptors are nowadays being coupled to antibodies. Indeed, the above examples have also strongly shown that purinoceptor- targeted small molecules and antibodies are intertwined in revealing the localization and function of these receptors. Commercially available antibodies provided by Alomone Labs are undeniably valuable tools in understanding and deciphering the specific purinergic receptor subtype in- volved, a task that most pharmacological tools has yet to show. The anti-P2Y receptor and the anti-P2X receptor antibodies enable the detection of the various receptors via Western blot analysis, immunohistochemistry, immunocy- tochemistry, as well as indirect or direct flow cytometry. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. References 1. Harden TK, Sesma JI, Fricks IP, Lazarowski ER (2010) Signalling and pharmacological properties of the P2Y receptor. Acta Physiol (Oxf) 199:149–160