Daniel:Notebook/RNAFISH/2015-2-25
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Linnarsson Protocol (Started 2/24/2015)
Buffer Prep
- Hybridization Buffer
- RNAse free water 5.3 mL
- SSC 20X 1 mL
- Dextran sulfate 2 mL
- Formamide 1 mL
- E coli tRNA 500 uL
- RVC 200 mM (warm to 37) 100 uL
- BSA 50 mg/mL 40 uL
Ethanol Precipitation
- Centrifuge at 4 C for 30 minutes
- Remove supernatant and add 750 uL chilled 75% EtOH
- Centrifuge at minutes at 4 C for 10 minutes
- Dry the pellet in the hood; protect from light
- Resuspend pellet in 15 uL TE buffer
Hybridization
- Prepare fresh paraformaldehyde 4% and keep at RT
- Fix sections with 4% paraformaldehyde for 15 minutes at RT
- Rinse sections twice with RT PBS
- Permeabilize with ethanol (70% EtOH, >90 minute incuabation at 4C)
- Rinse sections twice with RT PBS
- Prepare the wash buffer and let it sit at RT
- Warm hybridization buffer and warm it up to 37C
- Prepare hybridization reaction:
- hybridization buffer-100 uL; probe 2-10 uL
- Incubate o/n - 24 hours at 37C depending on tissue/thickness/etc.