PMID- 26437182
OWN - NLM
STAT- MEDLINE
DCOM- 20160916
LR  - 20181202
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 103
DP  - 2015 Sep 23
TI  - Rapid In Situ Hybridization using Oligonucleotide Probes on 
      Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome.
LID - 10.3791/53165 [doi]
LID - 53165
AB  - 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.
FAU - Shokry, Ibrahim M
AU  - Shokry IM
AD  - Ross University School of Veterinary Medicine.
FAU - Callanan, John J
AU  - Callanan JJ
AD  - Ross University School of Veterinary Medicine.
FAU - Sousa, John
AU  - Sousa J
AD  - Charles E. Schmidt College of Medicine, Florida Atlantic University.
FAU - Tao, Rui
AU  - Tao R
AD  - Charles E. Schmidt College of Medicine, Florida Atlantic University; 
      rtao@health.fau.edu.
LA  - eng
GR  - R15 DA029863/DA/NIDA NIH HHS/United States
GR  - R15DA029863/DA/NIDA NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PT  - Video-Audio Media
DEP - 20150923
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - 0 (Fixatives)
RN  - 0 (Oligonucleotide Probes)
RN  - 0 (Polymers)
RN  - 0 (RNA, Messenger)
RN  - 0 (Receptor, Serotonin, 5-HT2A)
RN  - 137497-17-7 (cyclophilin B)
RN  - 1HG84L3525 (Formaldehyde)
RN  - EC 1.17.1.8 (Dihydrodipicolinate Reductase)
RN  - EC 5.2.1.- (Cyclophilins)
RN  - KE1SEN21RM (N-Methyl-3,4-methylenedioxyamphetamine)
RN  - Y19UC83H8E (paraform)
SB  - IM
MH  - Animals
MH  - *Brain Chemistry
MH  - Cyclophilins/genetics
MH  - Dihydrodipicolinate Reductase/genetics
MH  - Fixatives/chemistry
MH  - Formaldehyde/chemistry
MH  - In Situ Hybridization/*methods
MH  - N-Methyl-3,4-methylenedioxyamphetamine/toxicity
MH  - *Oligonucleotide Probes
MH  - Paraffin Embedding/methods
MH  - Polymers/chemistry
MH  - RNA, Messenger/metabolism
MH  - Random Allocation
MH  - Rats
MH  - Receptor, Serotonin, 5-HT2A/genetics
MH  - Serotonin Syndrome/*genetics/metabolism
MH  - Tissue Fixation/methods
PMC - PMC4692616
MID - NIHMS849888
EDAT- 2015/10/06 06:00
MHDA- 2016/09/17 06:00
PMCR- 2017/09/23
CRDT- 2015/10/06 06:00
PHST- 2015/10/06 06:00 [entrez]
PHST- 2015/10/06 06:00 [pubmed]
PHST- 2016/09/17 06:00 [medline]
PHST- 2017/09/23 00:00 [pmc-release]
AID - 53165 [pii]
AID - 10.3791/53165 [doi]
PST - epublish
SO  - J Vis Exp. 2015 Sep 23;(103):53165. doi: 10.3791/53165.