Hybridization Chain Reaction Fluorescence In Situ Hybridization (HCR-FISH) in Ambystoma mexicanum Tissue

Area of Science:

• Regenerative Biology
• Molecular Biology
• Genomics

Background:

• In situ hybridization is crucial for visualizing mRNA in tissues.
• Fluorescent probes and signal amplification have advanced multiplexed RNA imaging.
• Axolotl regeneration provides a model for studying tissue repair and development.

Purpose of the Study:

• To present detailed protocols for multiplexed hybridization chain reaction fluorescence in situ hybridization (HCR-FISH).
• To enable mRNA quantification and imaging in regenerating axolotl tissue sections and whole developing limbs.
• To provide standardized methods for advanced RNA analysis in a key regenerative model.

Main Methods:

• Development of protocols for HCR-FISH staining and imaging.
• Implementation of cell segmentation techniques for analysis.
• Adaptation of methods for both tissue sections and whole-mount preparations.
• Application in regenerating axolotl tissues and developing limbs.

Main Results:

• Successful multiplexed RNA imaging in axolotl tissue sections.
• Quantification of mRNA levels through cell segmentation.
• Demonstration of whole-mount HCR-FISH for developing axolotl limbs.
• Establishment of comprehensive protocols for axolotl RNA analysis.

Conclusions:

• The presented HCR-FISH protocols are effective for multiplexed RNA analysis in axolotls.
• These methods facilitate detailed study of gene expression during regeneration and development.
• The protocols offer a valuable resource for researchers in regenerative medicine and developmental biology.