Nonradioactive, digoxigenin (DIG)-labeled cRNA probes with either sense or antisense orientation were synthesized by in vitro transcription using DIG labeling mix (Roche) according to the recommendations of the manufacturer. Probes were synthesized from cDNA clones encoding Fos purchased from Dharmacon (MMM1013-202760329). For fluorescent in situ hybridization (FISH), all solutions were prepared using RNase-free reagents and diethylpyrocarbonate (DEPC)-treated double deionzide water (ddH2O). All the brain sections were processed as described previously (Cho et al. 2016 (link)).
Protocol detail
In Situ Hybridization Using DIG-Labeled RNA Probes
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Brain
Cdna
Clones
Crna
Diethylpyrocarbonate
Digoxigenin
Fluorescent in situ hybridization
Rnase
Transcription
Corresponding Organization :
Other organizations : Harvard University
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Variable analysis
independent variables
- Probe type (sense or antisense orientation)
- MRNA expression (Fos)
- Use of RNase-free reagents and DEPC-treated double deionized water
- Brain sections processed as described previously (Cho et al. 2016)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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