Frozen tissue preparation for high-resolution multiplex histological analyses of human brain specimens.
Author(s): Shao F, Jiang W, Gao Q, Li B, Sun C, Wang Q, Chen Q, Sun B, Shen H, Zhu K, Zhang J, Liu C
Publication: J Neurooncol, 2017, Vol. 135, Page 21-28
PubMed ID: 28795278 PubMed Review Paper? No
Purpose of Paper
This paper compared the effects of preservation by snap-freezing, formalin fixation and paraffin embedding, and an optimized fixation protocol (that combined formaldehyde, OCT, and snap-freezing) on cellular morphology.
Conclusion of Paper
Cellular integrity was deteriorated in snap-frozen tissue, but fixation using an optimized procedure (that combined formaldehyde, OCT, and snap-freezing) resulted in cellular morphology staining that was comparable to that of formalin-fixed paraffin-embedded (FFPE) tissue specimens.
Studies
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Study Purpose
This study compared the effects of preservation by snap-freezing, formalin fixation and paraffin embedding, and an optimized fixation protocol on cellular morphology. Fresh glioma tissues were obtained during surgery and normal brain tissue was collected within 8 h postmortem from patients without detectable central nervous system (CNS) diseases. Tissues were diced and snap-frozen in liquid nitrogen, formalin-fixed and paraffin-embedded (FFPE), or preserved using an optimized protocol consisting of fixation in ice-cold 4% paraformaldehyde, dehydration in 30% sucrose, embedding with OCT, freezing on dry ice, and then storage at −80°C. Tissue sections (20 μm thick) were stained with PDGFRa (a membrane marker) and Olig2 (a nuclear marker) and detected by immunofluorescence with a confocal microscope or prepared for transmission electro-microscopy (TEM).
Summary of Findings:
Cellular integrity was deteriorated in snap-frozen tissue, as indicated by a disruption of subcellular localization of PDGFRa and Olig2, but fixation using the optimized procedure (that included paraformaldehyde and freezing in OCT) resulted in comparable cellular morphological staining to FFPE tissue. Further, TEM analysis revealed complete destruction of nuclei and organelles in snap-frozen tissues but these structures remained intact when preserved using the optimized fixation protocol.
Biospecimens
Preservative Types
- Formalin
- OCT
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Morphology Electron microscopy Morphology Laser-scanning confocal microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Snap frozen
Formalin (buffered)
Formaldehyde/snap-freezing