RNAscope technology provides a more precise method for multiplex fluorescent cellular level in situ hybridization. Mice were sacrificed by decapitation following quick anesthesia by isoflurane. Brains were removed, snap-frozen in isopentane at −40°C, and stored at −80°C. Frozen brains were sectioned in the coronal plane at −20°C in a cryostat microtome at 18 μm, mounted on Super Frost Plus slides, and stored at −80°C. The RNA Scope Fluorescent Multiplex Reagent kit (cat. no. 320850, Advanced Cell Diagnostics, Newark, CA, USA) was used for mRNA staining. Probes used for staining were: mm-Nr3c1-C1, mm-Fkbp5-C2 and mm-Nr3c2-C3. The staining procedure was performed according to manufacturer’s specifications as described previously (McCullough et al., 2018). Briefly, sections were fixed in 4% paraformaldehyde for 15 min at 4°C. Subsequently, brain sections were dehydrated in increasing concentrations of ethanol. Next, tissue sections were incubated with protease IV for 30 min at room temperature. Probes were hybridized for 2 h at 40°C followed by 4 hybridization steps of the amplification reagents 1 to 4. Next, sections were counterstained with DAPI (4′,6-diamidino-2-phenylindole), coverslipped and stored at 4°C until image acquisition. Sixteen-bit images of the dorsal hippocampus were acquired on a Leica SP8 confocal microscope using a 40x objective (n = 4 mice). For every individual marker, all images were acquired using identical settings for laser power, detector gain, and amplifier offset.

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