Lead contact
Materials availability
Data and code availability
RESOURCE AVAILABILITY
Primary hippocampal neuronal cell culture
Animals and animal housing
Human postmortem microarray analysis
EXPERIMENTAL MODEL AND SUBJECT DETAILS
In situ hybridization
Immunohistochemistry
Acute stress paradigm
Corticosterone assessment
Dexamethasone treatment
CORT, RU486, and spironolactone treatment
qPCR
Biotinylated oligoIP
Western blot analysis
Single-cell RNA sequencing analysis
METHOD DETAILS
QUANTIFICATION AND STATISTICAL ANALYSIS
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.