RNA Detection

3.5 Signal Detection
and Mounting

Detection of amplified in situ signal is dependent on the desired

chemistry and probe design. Here we describe the detection of

conjugated HRP-based green probe directed against microRNA-

101a and AP-based Fast Red probes designed to detectfosab

mRNA (Fig.4). Red and green signals are detected sequentially.

1. Add ~200μL of the freshly prepared Red working solution
   directly to the sections and incubate for 30 min at room tem-
   perature. Cover the entire humidified chamber with aluminum
   foil to keep the slides in the dark.


2. Tap slides on a stack of paper towels to remove as much of the
   detection solution as possible. Transfer slides directly into a
   Coplin jar with 1PBT buffer. Agitate by moving slides up and
   down during the 2 min wash. Repeat this wash for a total of
   three times.


3. Remove excess liquid by tapping slides on a stack of paper
   towels.


4. For detection of HRP-Green, add ~200μL of freshly prepared
   Green working solution to slides. Cover the humidified cham-
   ber and wrap with aluminum foil. Incubate for 10 min at room
   temperature.


5. Tap slides on a stack of paper towels to remove as much of the
   detection solution as possible.


6. Transfer slides directly into a Coplin jar with reverse osmosis
   water. Agitate by moving slides up and down during the 2 min
   wash. Repeat this wash for a total of three times.


7. Remove excess liquid by tapping slides on a stack of paper
   towels (seeNote 15).


8. Dry slides in a hybridization oven at 60C for 30 min. It is
   critical that slides are completely dry.


9. Immerse slides into 100% CitriSolv in a Coplin jar.

Fig. 4RNAscope ISH detection of microRNA-101 and fosab with RNAscope probes. Uninjured and regenerat-
ing zebrafish hearts were extracted and cryosectioned at 10μm and hybridized with either a negative control
or microRNA-101a (green) or fosab (red) probes. Scale bar¼ 50 μm

ISH Detection of MicroRNA and Target Gene mRNAs 205