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Hybridization.md

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Protocol for OligoSTORM hybridization

This version synchronized with our protocols paper Oligo STORM

Method

All steps are carried at room temperature, unless indicated otherwise. Conduct all RNaseA work such that RNase A does not contaminate work areas and equipment.

Sample fixation

  1. Prepare a sample of tissue culture cells adhered at desired confluency to a coverslip (polylysine coating of the coverslip may be necessary to ensure cells adhere properly, depending on the cell line).
  2. Gently rinse cells with 1X PBS by briefly immersing coverslip in a small petri dish containing the buffer and then placing into a fresh, dry petri dish.
  3. Immediately add enough freshly prepared fixation solution (5% PFA in PBS) to cover the cells and let stand for 10 minutes.
  4. Remove fixation solution and rinse the sample once or twice with 1X PBS.

Sample permeabilization

  1. Incubate fixed cells for 10 minutes in Permeabilization solution (0.5% Triton-X in PBS).
  2. Wash 2X in 1X PBS.
  3. Incubate for 5 minutes in 0.1 M HCl.
  4. Wash 3X for 1 minute in PBS.
  5. For DNA FISH only: incubate 1 hour at 37°C in RNaseA solution.
  6. Wash 3X for 1 minute in 2X SSC.
  7. Incubate for 35 minutes in 2X SSC + 50% vol/vol formamide and 0.1% Tween.
  8. Use sample directly in step or store at 4˚C. Coverslips may be stored at 4°C for several days.

Sample denaturation and hybridization (assuming one sample):

  1. Prepare hybridization mix on ice:
    • 1-5 µL of concentrated probe (~1000 ng/uL)
    • 20-70 µL of hybrdization buffer (see below).
  2. Add hybridization mix to a clean glass slide or a small petri dish.
  3. Gently tap coverslip of cells dry on a Kimwipe. Invert coverslip carrying the cells onto the solution on the glass slide.
  4. Seal edges of coverslip with rubber cement and allow the rubber cement to dry for 5 minutes.
  5. Denature the slides at 78°C-85°C for 3 minutes.
  6. Hybridize overnight at 42°C. If an air incubator is being used, place the sample in a humidified chamber.

Post-hybridization washes and preparation for imaging

  1. Pre-warm 2X SSCT solution to 42°C.
  2. Remove the coverslip and wash for 10 minutes each in 2X SSCT at 42°C.
  3. Wash in 2X SSC. Sample may be stored in 2X SSC at 4˚C for up to 2 weeks.

Mounting sample

  1. Make chambered slide: 1.1. Adhere two strips of double stick tape along both long edges of a standard slide. 1.2. Gently tap off excess fluid from coverslip and mount it on the doublestick tape (cells facing the slide).
  2. Add STORM imaging buffer to side of coverslip and allow channel to fill by capillary action.
  3. Seal coverslip to slide with nail polish.

Materials

All solutions should be prepared using distilled, deionized water (ddH2O). Take care to avoid nuclease contamination, particularly from DNases. The recipes provided here for stock solutions will support many samples, while the rest are intended for a single sample.

Sample fixation

  1. Coverslip #1.5 (Electron Microscopy Sciences, 72230-01).
  2. Tissue culture cell suspension.
  3. 1X phosphate buffered solution (PBS) pH 7.4 solution: Dilute a 10X PBS pH 7.4 stock solution (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4) ten-fold in ddH2O.
  4. Fixation solution: Combine 4 ml 10X PBS pH 7.4, 5 ml 32% (vol/vol) paraformaldehyde, and 31 ml ddH2O. Vortex vigorously to mix. Best to prepare fresh. Solution may be kept at RT.

Sample permeabilization

  1. Borohydride solution: 1 mg/ml sodium borohydride in dH2O, make fresh.
  2. 1X PBS.
  3. Permeabilization solution: 1X PBS + 0.5% (vol/vol) Triton X-100 (Sigma, T8787-100ML):
    • 10 ml 1X PBS + 50 µl Triton X-100. Always prepare fresh or filter sterilize
  4. Kimwipes.
  5. Ceramic rack.
  6. 0.1 N HCl (425 µl of 36% HCl in 50 ml = 0.1 M).
  7. RNaseA solution: 100 μg/ml RNaseA in PBS.
  8. 2X SSC solution (Ambion).
  9. Formamide (EMD Millipore, 344206-1L, or Amresco, 0606; see Note B4.1).
  10. 0.1% Tween-20 (Sigma, P1379-500ML).

Sample denaturation and hybridization:

  1. Hybridization buffer: 50% formamide, 2X SSC, 0.1% Tween-20, 10% Dextran Sulfate.
  2. Oligopaint oligos (assuming a single sample): ~100 pmol of Oligopaint oligos per 100 kb of genomic target.
  3. Reporter-labeled secondary probe: 100 µM solution.
  4. Heat source for denaturation.
  5. Hybridization incubator.
  6. Humidified chamber.
  7. Glass slide.

Post-hybridization washes

  1. 2X SSCT solution: 2X SSC + 0.1% vol/vol Tween 20.
  2. 2X SSC.

Mounting sample

  1. Standard glass slide (VWR, 48311-601).
  2. STORM imaging buffer: * 400 µl of 2X SSC. * 100 µl of 50% glucose. * 10 µl of GLOX buffer: 175 µl 2X SSC, 10 mg glucose oxidase powder (Sigma, G2133-250KU), and 25 µl catalase at 16 mg/ml (Sigma, C40-500MG). Can be stored up to 1 week at 4°C). * 5 µl β-mercaptoethanol (Sigma, 63689-25ML-F).
  3. Nail polish (Electron Microscopy Sciences, 72180).