HCR DNA-FISH¶
Prepare necessary buffers as described in FISH buffer recipes Expected time: 2 days
Fixation¶
Steps 1-5 of Immunofluorescent Staining
Hybridization¶
Preheat Hybridization Buffer (HYB) to 37 degrees Celsius
Incubate cells at room temperature for 30 minutes in 500 uL of 20% glycerol in PBS
Flash freeze the cells
Place coverslips in liquid nitrogen for 30 seconds
Remove and let thaw for 1 minute
Warning
Liquid Nitrogen can shatter the coverslip; be careful when freezing
Incubate cells at RT for 20 mins in 500 uL of 20% glycerol in PBS
Repeat Step 4
Incubate cells at RT for 5 mins in 500 uL of .1 N HCl
Rinse cells at RT with 500 uL of 2X SSC
Repeat x2
Incubate cells at 37 degrees C in 500 uL of HYB for 30 mins
Remove HYB and add 100 uL of probe+HYB mixture
Incubate at 78 degrees C for 5 mins
Incubate overnight at 37 degrees C in a humid environment
Probe Preperation¶
Preheat Hybridization Buffer (HYB) to 37 degrees Celsius
Dilute probe stock with HYB to 10 pmol per 100 uL per reaction
Heat probe+HYB mixture at 70 degrees C for 5 mins
Chill on ice until use
Post-Hybridization¶
Preheat Hybridization Buffer (HYB) to 37 degrees Celsius
Preheat Probe Wash Buffer to 37 degrees C
Remove probe+HYB, then wash with 500 uL of Probe Wash Buffer
Repeat
Wash at RT with 500 uL of 5X SSCT for 5 mins
Repeat
Wash at RT with 500 uL of PBS for 5 mins
Proceed to imaging