Effect of formalin-fixation and paraffin-embedded tissue storage times on RNAscope in situ hybridization signal amplification.
J Vet Diagn Invest
; 36(4): 498-505, 2024 Jul.
Article
in En
| MEDLINE
| ID: mdl-38650110
ABSTRACT
RNAscope in situ hybridization (ISH) detects target RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. Protocols suggest that prolonged FFPE storage and formalin fixation may impact signal detection, potentially limiting the utility of RNAscope ISH in retrospective studies. To develop parameters for RNAscope use with archived specimens, we evaluated the effect of formalin-fixation time by measuring the signal of a reference gene (16srRNA) in selected tissues fixed in 10% neutral-buffered formalin for 1, 2, 3, 5, 7, 10, 14, 21, 28, 60, 90, 180, and 270 d. The signal intensity and percent area of signal decreased after 180 d. Tissues had detectable signal at 180 d but not at 270 d of formalin fixation. To assess target detection in paraffin blocks, we qualitatively compared the signal of canine distemper virus (CDV) antigen via immunohistochemistry and CDV RNA via RNAscope ISH in replicate sections from blocks stored at room temperature for 6 mo, 1, 3, 6, 8, 11, 13, and 15 y; RNA was detected in FFPE tissues stored for up to 15 y. Our results demonstrate that RNAscope ISH can detect targets in tissues with prolonged paraffin storage intervals and formalin-fixation times.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Tissue Fixation
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Paraffin Embedding
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In Situ Hybridization
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Formaldehyde
Limits:
Animals
Language:
En
Journal:
J Vet Diagn Invest
/
J. vet. diagn. invest
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Journal of veterinary diagnostic investigation
Journal subject:
MEDICINA VETERINARIA
Year:
2024
Document type:
Article
Affiliation country:
Country of publication: