HCR RNA-FISH¶
Prepare necessary buffers as described in FISH buffer recipes Expected time: 3 days
Note
All spins are performed at ~500 rcf for 4 min. Our centrifuge follows RCF = 1e-4*[rpm]^2 + 4e-2*[rpm] - 6e1, where 2200 rpm = 512 rcf. It is recommended to perform all spins at 4°C once the cells have been fixed to prevent pellet loss.
Warning
Both PFA (used in the fixation) and Formamide (used in the hybridization buffers) are hazardous, so all steps should be performed in the fume hood
Solution Detection Stage¶
Steps 1-5 of Immunofluorescent Staining
Add 200 uL of probe hybridization buffer for 30 min at 37 degrees C
Prepare probe solution by adding 2 uL of 1 uM stock to 500 uL of probe hybridization buffer at 37 degrees C
Remove pre-hybridization solution and add 200 uL of probe/hybrid buffer solution
Incubate samples overnight (12-16 h) at 37 degrees Celsius
Remove excess probes by washing 1x 15 min with 200 uL of probe wash buffer at 37 degrees Celsius
Wash samples 1x 5 min with 200 uL of 5X SSCT at room temperature
Solution Amplification Stage¶
Pre-amplify samples in 500 uL of amplification buffer for 30 min at room temperature While waiting, begin Hairpin Preparation:
Prepare 30 pmol of hairpin h1 and h2 by snap cooling 10 uL of 3 uM stock at 95 degrees Celsius for 90 seconds
Cool hairpins in the dark at room temperature for 30 minutes
Prepare hairpin solution by adding h1 and h2 hairpins to 500 uL amplification buffer
Remove pre-amplification solution and 200 uL of the hairpin/amplification buffer solution
Incubate samples overnight (12-16 h) in the dark at room temperature
Remove excess hairpins by washing with 200 uL of 5X SSCT at room temperature for:
1x 30 min
1x 5 min
Store at 4 degrees C in the dark until imaging