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

Take fixed brain sections from a rat with serotonin syndrome, which leads to altered serotonin receptor RNA synthesis in the neurons.

Dehydrate the tissue using alcohol. Apply a pretreatment solution to deactivate endogenous hydrolyzing enzymes, preventing their interaction with detection reagents.

Treat with a boiling buffer to break fixation-induced crosslinks, exposing the target RNAs.

Dehydrate the tissue, draw a hydrophobic barrier, and incubate it with a solution to permeabilize cellular membranes.

Add oligonucleotide probes and incubate, allowing the probes to hybridize with the target RNAs.

Wash to remove unbound probes, then incubate with amplification reagents containing preamplifier, amplifier, and enzyme-conjugated label probes that bind to the oligonucleotide probes.

Incubate with chromogenic substrates that get hydrolyzed by the enzyme-conjugated probes, producing visible color.

Treat with xylene to increase tissue transparency, add a mounting medium, and apply a coverslip.

Under a microscope, colored spots in the tissue help quantify target RNA expression.