We performed smFISH as described previously (71 (link)). Briefly, inner ear tissues at P1 were dissected out for cryosection. The slices were treated with 10 μg/mL Proteinase K solution at room temperature for 5 min, followed by 4% PFA to stop the Proteinase K reaction, and then incubated with the digoxigenin-labeled Atoh1 probe; lastly, a tyramide signal amplification kit (catalog NEL753001KT, PerkinElmer) was used to visualize Atoh1 mRNA. After completing the aforementioned procedure, the slices were incubated with anti-Myo7a antibody overnight at 4 °C.
Protocol detail
Atoh1 Expression in Inner Ear Tissue
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Corresponding Organization :
Other organizations : Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Institute of Genetics and Developmental Biology, Shanghai Center for Brain Science and Brain-Inspired Technology
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Variable analysis
independent variables
- Proteinase K treatment (10 μg/mL at room temperature for 5 min)
- Atoh1 mRNA expression (visualized using a tyramide signal amplification kit)
- Tissue type (inner ear tissues at P1)
- Cryosection preparation
- PFA fixation to stop Proteinase K reaction
- Incubation with digoxigenin-labeled Atoh1 probe
- Incubation with anti-Myo7a antibody overnight at 4 °C
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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