title: Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome.
abstract: 3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.
authors.affiliation: Ross University School of Veterinary Medicine.
authors.forename: Ibrahim M
authors.initials: IM
authors.lastname: Shokry
authors.affiliation: Ross University School of Veterinary Medicine.
authors.forename: John J
authors.initials: JJ
authors.lastname: Callanan
authors.affiliation: Charles E. Schmidt College of Medicine, Florida Atlantic University.
authors.forename: John
authors.initials: J
authors.lastname: Sousa
authors.affiliation: Charles E. Schmidt College of Medicine, Florida Atlantic University; rtao@health.fau.edu.
authors.forename: Rui
authors.initials: R
authors.lastname: Tao
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D021983:Cyclophilins / Q000235:genetics; D050746:Dihydrodipicolinate Reductase / Q000235:genetics; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D017403:In Situ Hybridization / Q000379:methods*; D018817:N-Methyl-3,4-methylenedioxyamphetamine / Q000633:toxicity; D015345:Oligonucleotide Probes; D016612:Paraffin Embedding / Q000379:methods; D011108:Polymers / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D011897:Random Allocation; D051381:Rats; D044402:Receptor, Serotonin, 5-HT2A / Q000235:genetics; D020230:Serotonin Syndrome / Q000235:genetics* / Q000378:metabolism; D016707:Tissue Fixation / Q000379:methods
chemical_list: D005404:Fixatives; D015345:Oligonucleotide Probes; D011108:Polymers; D012333:RNA, Messenger; D044402:Receptor, Serotonin, 5-HT2A; C081786:cyclophilin B; D005557:Formaldehyde; D050746:Dihydrodipicolinate Reductase; D021983:Cyclophilins; D018817:N-Methyl-3,4-methylenedioxyamphetamine; C003043:paraform