Double-label nonradioactive in situ hybridization for the analysis of chemokine receptor expression in the central nervous system.
Chemokines are a family of mainly-secreted proteins, traditionally associated with regulation of leukocyte trafficking during host defense and pathological immune/inflammatory reactions. All chemokines signal to G protein-coupled receptors. Recent studies show that chemokines and their receptors are also expressed by neuroepithelial cells, and govern developmental, physiological and pathological processes through actions towards these cells, as well as infiltrating and resident hematopoietic cells. Understanding chemokine action at the tissue level therefore requires defining which cells express chemokine receptors. At a first level of approximation (and lacking appropriate immunohistochemical reagents) this determination can be made by in situ hybridization (ISH), which localizes mRNA expression for chemokines and their receptors at the cellular level. Here we provide a protocol for ISH and demonstrate its application for localizing mRNA encoding two chemokine receptors, CXCR4 and CXCR7 in murine CNS tissues.
Li
Meizhang
M
Neuroinflammation Research Center, Department of Neurosciences, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
Ransohoff
Richard M
RM
D000818:Animals; D002490:Central Nervous System / Q000378:metabolism*; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics; D054387:Receptors, CXCR / Q000235:genetics; D019718:Receptors, CXCR4 / Q000235:genetics; D019707:Receptors, Chemokine / Q000235:genetics*
C501897:Cmkor1 protein, mouse; D012333:RNA, Messenger; D054387:Receptors, CXCR; D019718:Receptors, CXCR4; D019707:Receptors, Chemokine