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Protocol for Single-Molecule Fluorescence In Situ Hybridization for Intact Pancreatic Tissue
Lydia Farack1, Shalev Itzkovitz1
1Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.
STAR Protocols
|October 28, 2020
Summary
We optimized single-molecule fluorescence in situ hybridization (smFISH) for intact pancreas tissue. This enhanced protocol allows sensitive single mRNA visualization in the pancreas, overcoming previous limitations.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Standard single-molecule fluorescence in situ hybridization (smFISH) protocols are effective for most tissues but not the intact pancreas.
- Previous methods failed to provide sensitive single mRNA visualization in pancreatic tissue.
Purpose of the Study:
- To develop and optimize a single-molecule fluorescence in situ hybridization (smFISH) protocol specifically for the intact pancreas.
- To enable sensitive visualization of single mRNA molecules within pancreatic tissue.
Main Methods:
- Adaptation of a generic tissue smFISH protocol.
- Increased mRNA denaturation time from 5 minutes to at least 3 hours.
- Increased formamide concentration from 10% to 30%.
Main Results:
- Achieved sensitive single mRNA visualization in the intact pancreas.
- The optimized protocol yields results comparable to standard smFISH in other tissues.
- Successfully overcame limitations of previous smFISH applications in pancreatic tissue.
Conclusions:
- The modified smFISH protocol is effective for sensitive mRNA detection in the intact pancreas.
- This protocol advances molecular analysis of pancreatic tissue.
- Further details are available in Farack et al., 2018 and 2019.
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