- Remove slides from Coplin jar and gently tap on a stack of
paper towels to remove excess PBS. It is not necessary to
completely dry slides before proceeding. - With slides on the bench, add 500μL of 3% hydrogen peroxide
solution to each slide. Incubate for 10 min at room tempera-
ture (seeNote 10). - Rinse twice with filtered water in Coplin jars. It is best to
transfer the slides from one Coplin jar to another instead of
decanting the water. - With a pair of blunt forceps, transfer the slides into the boiling
antigen retrieval buffer. Incubate for 10 min (seeNote 11). - Transfer the slides directly into a clean Coplin jar with water.
Gently agitate by moving slides up and down four times. - Repeat this wash by moving slides into a clean Coplin for a total
of three washes. - Wash slides in 100% ethanol. Gently move slides up and down
five times. Transfer slides to a stack of paper towels and allow to
air-dry ~2 min. - Redraw a barrier around the tissues with the hydrophobic pen.
It is important to ensure that there are no gaps in the barrier.
3.4 Hybridization Hybridization and amplification of the in situ RNA signal is
performed in accordance with ACD suggested protocol with few
modifications. Two critical factors during this process are: (1) main-
taining hybridization temperature at 40C and (2) keeping slides
moist between washes and/or incubation steps (seeNote 12).
- Place slides directly onto the serological pipettes within the
humidified chamber and add 4–6 drops of ACD Protease Plus
solution onto the sections. Add more drops if necessary to
cover all sections (seeNote 13). - Cover the chamber with the Tupperware lid and close tightly.
Incubate at 40C for 30 min (seeNote 3; Fig.3). - Wash slides with water in a Coplin jar. Provide gently agitation
by moving slides up and down four times. Repeat wash for a
total of three washes. - Conjugated RNA probe is provided in a dropper bottle from
ACD. Add a sufficient amount of the probe mixture to cover all
sections on slides (seeNote 13). Incubate at 40C for 2 h. If
necessary, this incubation may be extended to up to 6 h (see
Note 14). - Wash slides with 1PBT buffer in Coplin jars at room temper-
ature. Agitate by moving slides up and down during the 2 min
wash. Repeat this wash for a total of three times.
ISH Detection of MicroRNA and Target Gene mRNAs 203