Home › Cell Biology › Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation
Cell Biology JoVE (Open Access) Citable · DOI

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

DOI: 10.3791/55885-v
What you'll learn
  • ✓ Prepare tissues using non-cross-linking, formalin-free fixative for preservation
  • ✓ Perform HER2 FISH on NCFPE specimens with post-fixation protocol
  • ✓ Compare FISH signal quality between NCFPE and standard FFPE samples
  • ✓ Analyze HER2 status using combined morphologic and molecular data
Protocol

Fluorescence in-situ hybridization (FISH) is often required in combination with histopathology and molecular diagnostics for selection of therapy in personalized medicine. A novel non-cross-linking, formalin-free tissue fixative that allows high quality morphologic, molecular and FISH analyses from the same specimen by addition of a post-fixation step before FISH is presented.

Difficulty
advanced
Total time
~4–6 hours per tissue batch (excluding initial fixation time of 12–24 hours)
Biosafety
BSL-1

Steps

1
Fixate tissue specimens in non-cross-linking formalin-free fixative

Place freshly isolated tissue samples into the novel NCFPE fixative solution, allowing preservation of morphologic and molecular integrity without formaldehyde-induced cross-linking.

▶ 01:28
2
Apply post-fixation step before HER2 FISH protocol

Perform post-fixation treatment on NCFPE specimens to optimize tissue permeability and signal detection before fluorescence in-situ hybridization.

▶ 03:54
3
Execute HER2 FISH assay on prepared tissue sections

Conduct fluorescence in-situ hybridization targeting HER2 locus on post-fixed NCFPE tissue sections following standard FISH protocol modifications.

▶ 03:54
4
Analyze FISH signals and compare NCFPE versus FFPE results

Evaluate HER2 FISH signal intensity and morphologic quality, correlating NCFPE and standard FFPE specimens alongside real-time reverse transcription PCR validation.

▶ 07:37
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