HCR DNA-FISH

Prepare necessary buffers as described in FISH buffer recipes Expected time: 2 days

Fixation

  1. Steps 1-5 of Immunofluorescent Staining

Hybridization

  1. Preheat Hybridization Buffer (HYB) to 37 degrees Celsius

  2. Incubate cells at room temperature for 30 minutes in 500 uL of 20% glycerol in PBS

  3. Flash freeze the cells

    1. Place coverslips in liquid nitrogen for 30 seconds

    2. Remove and let thaw for 1 minute

Warning

Liquid Nitrogen can shatter the coverslip; be careful when freezing

  1. Incubate cells at RT for 20 mins in 500 uL of 20% glycerol in PBS

  2. Repeat Step 4

  3. Incubate cells at RT for 5 mins in 500 uL of .1 N HCl

  4. Rinse cells at RT with 500 uL of 2X SSC

    1. Repeat x2

  5. Incubate cells at 37 degrees C in 500 uL of HYB for 30 mins

  6. Remove HYB and add 100 uL of probe+HYB mixture

  7. Incubate at 78 degrees C for 5 mins

  8. Incubate overnight at 37 degrees C in a humid environment

Probe Preperation

  1. Preheat Hybridization Buffer (HYB) to 37 degrees Celsius

  2. Dilute probe stock with HYB to 10 pmol per 100 uL per reaction

  3. Heat probe+HYB mixture at 70 degrees C for 5 mins

  4. Chill on ice until use

Post-Hybridization

  1. Preheat Hybridization Buffer (HYB) to 37 degrees Celsius

  2. Preheat Probe Wash Buffer to 37 degrees C

  3. Remove probe+HYB, then wash with 500 uL of Probe Wash Buffer

    1. Repeat

  4. Wash at RT with 500 uL of 5X SSCT for 5 mins

    1. Repeat

  5. Wash at RT with 500 uL of PBS for 5 mins

  6. Proceed to imaging