In situ hybridization analysis of chemokines and chemokine receptors in the central nervous system.
The expression of a number of chemokines and chemokine receptors by cells resident in normal and pathological central nervous system (CNS) tissue has been characterized by in situ hybridization techniques. As a result, our understanding of the role of this cytokine family in neurobiology has been enhanced greatly. Specific methods for detecting chemokine and chemokine receptor mRNAs in situ vary with the number of these genes that have been characterized and encompass approaches widely utilized by other investigators characterizing cell-specific gene expression patterns. We describe methods that our laboratory has used successfully in characterizing chemokine and chemokine receptor expression in the CNS, focusing on protocols that utilize radiolabeled in vitro-transcribed riboprobes for detecting these transcripts. Because general dye-based histological staining methods do not readily differentiate astrocytes and microglia, specific immunohistochemical protocols are required for definitive localization of gene expression to these glial cell types. As such, methods that are compatible with the in situ hybridization procedure are included for staining astrocytes and microglia.
Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, FL 32610-0267, USA. harrison@pharmacology.ufl.edu
Jeffrey K
JK
Harrison
Defang
D
Luo
Wolfgang J
WJ
Streit
D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D048588:Benzoxazines; D015152:Blotting, Northern / Q000379:methods; D002490:Central Nervous System / Q000378:metabolism*; D018925:Chemokines / Q000096:biosynthesis*; D004396:Coloring Agents / Q000494:pharmacology; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D009457:Neuroglia / Q000473:pathology; D010078:Oxazines / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D019707:Receptors, Chemokine / Q000096:biosynthesis*; D014158:Transcription, Genetic
D048588:Benzoxazines; D018925:Chemokines; D004396:Coloring Agents; D010078:Oxazines; D012333:RNA, Messenger; D019707:Receptors, Chemokine; C028911:cresyl violet