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Botany JoVE (Open Access) Citable · DOI

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

DOI: 10.3791/1205-v
What you'll learn
  • ✓ Perform DIG in situ hybridization on plant tissues with minimal radioactive handling
  • ✓ Optimize RNase and proteinase K treatment for diverse plant species
  • ✓ Prepare and section plant samples for hybridization analysis
  • ✓ Troubleshoot protocol adjustments for Norway spruce and other plant tissues
Protocol

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

Difficulty
advanced
Total time
~3-4 days (including fixation, embedding, sectioning, and hybridization)
Model organism
Norway spruce (Picea abies); applicable to multiple plant species
Biosafety
BSL-1

Steps

1
Fix and embed plant tissue samples

Prepare plant tissues by fixation and embedding in appropriate medium. This foundational step preserves tissue morphology and enables sectioning for subsequent hybridization analysis.

▶ 00:45
2
Section embedded samples for analysis

Cut thin sections from embedded plant tissue using standard sectioning equipment. Prepare slides with sections ready for in situ hybridization treatment.

▶ 01:41
3
Perform DIG in situ hybridization

Apply the modified DIG hybridization protocol with adjusted RNase and proteinase K concentrations optimized for plant species. Include probe hybridization, washing, and detection steps to visualize target sequences.

▶ 06:16
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