In Situ Hybridization Using Oligonucleotide Probes to Study RNA Expression in Rat Brain Sections

Abstract

Source: Shokry, I. M., et al. Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome. J. Vis. Exp. (2015).

This video demonstrates the detection of serotonin receptor RNA synthesis using oligonucleotide probes in rat brain tissue affected by serotonin syndrome. The protocol includes tissue pretreatment, probe hybridization, and enzymatic detection to visualize and quantify serotonin receptor RNA expression under a microscope.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Preparation

1. Tissue preparation and sectioning
   1. Assign rats randomly into three groups: fresh/saline (SAL; 0.9% NaCl), prefixed/SAL and prefixed/3,4-methylenedioxymethamphetamine (MDMA; see details in table 1).
   2. Administer three doses of 1 ml/kg SAL and 10 mg/kg MDMA intraperitoneally, respectively, to the SAL and MDMA groups at 2 hr interval. Allow animals to survive the treatment for 6 days.
   3. On day 7, administer 100 mg/kg ketamine in comb....

Results

Table 1. Experimental design

	Fresh	Paraformaldehyde-prefixeda
	SALb	SALb	MDMAc
Target probe (htr2a)	√	√	√
Positive probe (ppiB.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RNAscope Negative Control Probe-DapB	Advanced cell diagnostic, INC	310098
RNAscope Pretreat 4	Advanced cell diagnostic, INC	320046
RNAscope 2.0 HD Reagent Kit - Red	Advanced cell diagnostic, INC	310036
RNAscope Probe - Rn-Ppib	Advanced cell diagnostic, INC	313921
Rat Htr2a	Advanced cell diagnostic, INC	300031
Cryostat	Leica	CM 1850
Horizontal shaker	VWR	88032-088
Hybridization oven	Thermo Fisher Scientific	222000
Superfrost Plus microscope slide	Fisher Scientific	12-550-15
Hydrophobic pen	Vector	H-4000
Microscope	Olympus	Provis AX70