RNAscope in situ hybridization

FIXATION (4°C)

Immerse in 4% PFA for 15 MIN at 4°C

Make sure there is enough ethanol ready for the next steps

DEHYDRATION (ROOM TEMPERATURE)

50% EtOH for 5 MIN

70% EtOH for 5 MIN

100% for 5 MIN Use ethanol from the bottle labeled “one use”

100% fresh for 5 MIN. Dispose of this solution into “one use” EtOH bottle

HYDROPHOBIC BARRIER (ROOM TEMPERATURE)

Air dry for 5 MIN

Draw barrier (3 times), let dry for 1 MIN

Take out Pretreat 4 from the deli fridge during this time to warm to room temp

PRE-TREAT (ROOM TEMPERATURE)

Place slides in Rack and add drops of Pretreat 4

Incubate for 30MIN at ROOM TEMP

During this time, prepare PROBES and Wash Buffer

PROBE PREPARATION: Use DNAse free tubes!

Warm probes for 10 MIN at 40°C in water bath then cool to ROOM TEMP

Spin down C2 and C3 probes

Mix 1:50 for C2/3 : C1     Invert tube to mix and microfuge to collect to bottom of tube.

AFTER PRETREAT:

Tap off liquid from slide and wash in 1X PBS 

Move rack up and down in PBS solution 3-5 times

Repeat again with 1X PBS (Can stay in PBS for up to 15 minutes)

Important: in next step, add probe to one slide at a time while leaving remaining slides in PBS. 

PROBE INCUBATION 40°C

Tap off liquid and quickly add drops of probe

Place in oven for 2 HOURS at 40°C

Before the end of incubation, take out detection kit reagents (amplification solutions) to warm up to room temp

Prepare or check that there is enough wash buffer (50x stock solution => 1x will be 40mL into 2L MilliQ water)

Remove slide rack from tray, tap liquid off slides and place into 1X Wash Buffer (ONE AT A TIME)

Replace tray into incubator to keep warm.

Wash in 1X Wash Buffer for 2 MIN at ROOM TEMP while agitating

Repeat again in fresh 1X Wash Buffer

AMPLIFICATION (INCUBATIONS AT 40°C)

Hybridize Amp 1: add drops one at a time (Incubate 30 MIN at 40°C)

Wash for 2 MIN w/ 1X Wash Buffer at ROOM TEMP w/ agitation

Repeat wash (then keep wash buffer in container for next wash)

Hybridize Amp 2: add drops (Incubate 15 MIN at 40°C)

Quickly “rinse” slides in old wash buffer from last step, then

Wash for 2 MIN w/ fresh 1X Wash Buffer at ROOM TEMP w/ agitation

Repeat wash (keep old wash buffer)

Hybridize Amp 3: add drops one at a time (Incubate 30 MIN @ 40°C)

“Rinse” then wash for 2 MIN w/ fresh 1X Wash Buffer at ROOM TEMP w/ agitation

Repeat wash (keep old wash buffer)

Hybridize Amp 4: add drops (Incubate for 15 MIN at 40°C)

“Rinse” then wash for 2 MIN w/ fresh 1X Wash Buffer at ROOM TEMP w/ agitation

Repeat wash

COUNTERSTAIN AND MOUNT

Tap off liquid and add DAPI, incubate for 30 SECONDS at ROOM TEMP

Remove DAPI and immediately drop mounting medium onto tissue. Coverslip and store in the dark at 4⁰C