Daniel:Notebook/RNAFISH/2015-2-24: Difference between revisions

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>Djacobse
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==Dye Coupling==
==Dye Coupling==


*GAD1-488
*FOXP2-488
*FOXP2-594
*GAD1-594


#Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
#Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature

Revision as of 18:39, 24 February 2015

Linnarsson Protocol

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I'll be starting work today on the Linnarsson lab protocol for doing RNA FISH on tissue sections. The protocol is given to me from Simone, one of their main RNA FISH researchers, who visited our lab in Dec 2014/Jan 2015.

Buffer Prep

The protocol uses several solutions that are prepared beforehand and aliquoted for later use. Most of these need to only be done once.

  • E Coli tRNA 20 mg/mL
    • tRNA 100 mg
    • RNAse free H2O 2.5 mL
    • aliquot into 500 uL increments and store at -20C
  • DAPI stock solution
    • RNAse free water 500 uL
    • DAPI powder 10 mg
    • aliquot into 5 20 uL increments and store at -20C
    • store the remainder at -20C
  • DAPI ready to use
    • DAPI stock (20 mg/mL) 10 uL
    • nfH2O 990 uL
    • Store at 4C use within 1 month
  • 70% Ethanol
    • 700mL absolute EtOH
    • 300 mL ddi water
  • Ribonucleoside Vanadyl complex (200mM)
    • Incubate the purchased solution in a water bath at 65C for 10 minutes
    • Aliquot in 100 uL and store up to 4 months at -20C
  • BSA 50 mg/mL
    • Came as 1 mL of 50 mg/mL, not going to split it

NOTES:

  • I need fresh formamide and fresh paraformaldehyde 16% w/v I will order both of these
  • For now, I am using less fresh bottles

Dye Coupling

  • FOXP2-488
  • GAD1-594
  1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
  2. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
  3. Denature samples for 5 minutes at 95C, then snap cool using ice box
  4. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
    1. dye cannot be saved for later use. Use immediately!
  5. Add 2 uL dye to sample
  6. Incubate in the dark for 1 hour
  7. Add 10 uL 3M NaOAc and 80 uL nfH20 to sample
  8. Centri-Sep column purification after incubation
    1. Use TE Buffer

Hybridization

  1. Prepare fresh paraformaldehyde 4% and keep at RT
  2. Fix sections with 4% paraformaldehyde for 15 minutes at RT
  3. Rinse sections twice with RT PBS
  4. Permeabilize with ethanol (70% EtOH, >90 minute incuabation at 4C)
  5. Rinse sections twice with RT PBS
  6. Prepare the wash buffer and let it sit at RT
  7. Warm hybridization buffer and warm it up to 37C
  8. Prepare hybridization reaction:
    1. hybridization buffer-100 uL; probe 2-10 uL
  9. Incubate o/n - 24 hours at 37C depending on tissue/thickness/etc.