Daniel:Notebook/RNAFISH/2014-8-31
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Trial Run (Started 08/29/2014)
Dye Labeling
Sample Matrix
Sample | GAD1 | FOXP2 | RBFOX3 | CUX2 |
Dye | Alexa-488 | Alexa-546 | Alexa-594 | Alexa-647 |
ng/uL | 512 | 558 | 568 | 604 |
uL added | 10 | 10 | 10 | 10 |
ug added | 5.1 | 5.6 | 5.7 | 6.0 |
- Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
- Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
- Denature samples for 5 minutes at 95C, then snap cool using ice box
- Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
- dye cannot be saved for later use. Use immediately!
- Add 2 uL dye to sample
- Incubate in the dark for 1 hour
- Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
- Centri Sep purification after incubation
Labeling Results
Sample | ng/uL ssDNA | pmol/uL dye | pmol/uL ssDNA | dye:probe | bp:dye |
GAD1-488 | 55.7 | 9.2 | 8.4 | 1.1 | 18 |
FOXP2-546 | 67.7 | 13.7 | 10.3 | 1.3 | 15 |
RBFOX3-594 | 68.3 | 13.8 | 10.3 | 1.3 | 15 |
CUX2-647 | 59.8 | 12.3 | 9.1 | 1.4 | 15 |
Great labeling results, and each sample has ~50 uL to work with.
Pretreatment
Sample Matrix
uL GAD1-488 (pmol) | uL FOXP2-546 (pmol) | uL RBFOX3-594 (pmol) | uL CUX2-647 (pmol) | |
Sample A | 25 (211) | 0 | 25 (258.7) | 0 |
Sample B | 0 | 25 (256.4) | 0 | 25 (226.5) |
Sample A is the sample closer to the label, sample B is the sample further from the label.
- Remove slides from 4C, let them thaw and air-dry at RT for 30–60 min
- Re-hydrate sections in 10 mM sodium citrate buffer for 5 min
- Transfer slides into a Coplin jar with prewarmed (80°C) 10 mM sodium citrate buffer in a water bath and incubate there for 5–25 min
- Remove the Coplin jar from the water bath and let it cool down at RT
- Incubate the slides in 2× SSC for 5 min
- Transfer slides into 50% formamide in 2× SSC and incubate for at least 1 h
Hybridization
- Remove slide from the formamide and wipe the liquid from the back of the slide and from the face side of the slide
- Avoid marked section area
- Prepare glass hybridization chambers as follows:
- Roll a small piece of mounting putty (Blu Tack) into a snake.
- Wrap the mounting putty around tissue section, covering 3 sides
- Size of the chamber depends on a section size: chamber should fit to the section size as closely as possible in order to save DNA probe
- Place coverslip over putty, press gently to seal the coverslip to the putty
- Load the probe under chamber from its open side so that probe fills in the whole space above the section
- Seal the chamber with rubber cement
- It is important that rubber cement covers the entire chamber periphery or even the whole chamber
- Let the rubber cement dry at RT
- From this step, protect slide from light
- Place slide on a hot-block (or water bath) with temperature 45°C for 2 h in order to allow infiltration of the section with the probe
- Place slide on a hot-block at 80°C for 5 min for the probe and section denaturation of DNA
- Hybridize slide for 3 days at 37°C in a waterbath or temperature controlled oven; Continued 9/3/2014