Decreased D2-40 immunoreactivity in stored paraffin sections and methods for preserving it.
Author(s): Sasaki T, Kawabata Y, Suzuki N, Tanaka H, Sano M, Kato S, Takemura A, Unno N, Kojima T, Sato K
Publication: Biotech Histochem, 2014, Vol. 89, Page 412-8
PubMed ID: 24939609 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine if antigenicity of lymphatic endothelial markers is affected by storage of formalin-fixed paraffin-embedded (FFPE) slide-mounted tissue sections at different temperatures (room temperature, 4°C, -30°C) for up to 16 weeks.
Conclusion of Paper
Podoplanin immunostaining using the D2-40, but not the NZ-1, antibody was adversely affected by storage of slide-mounted FFPE sections at room temperature for 4 weeks in skin scalp specimens and 8 weeks for hair follicle and lymph node specimens. These storage-induced effects were attenuated by FFPE slide storage at temperatures lower than ambient, as FFPE slides stored for 8 weeks at 4°C or -30°C displayed stable podoplanin immunostaining with the D2-40 antibody in scalp skin specimens. The presence of silica gel during FFPE slide storage had no discernible effect on antigenicity. LYVE-1 immunostaining remained stable in FFPE sections stored at room temperature for up to 16 weeks.
Studies
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Study Purpose
The purpose of this study was to determine if the antigenicity of the lymphatic endothelial markers podoplanin and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1) is affected by storage of formalin-fixed paraffin-embedded (FFPE) slide-mounted tissue sections at different temperatures for up to 16 weeks. Four lymph node specimens and four scalp specimens (containing both skin and hair follicles) were obtained at autopsy and fixed in 4% paraformaldehyde in phosphate buffer (0.01 mol/L, pH 7.4) for 16 h at 4°C then processed and embedded in paraffin. FFPE sections (3 µm) were mounted on MAS-coated slides and stored in a sealed box protected from light at room temperature for 2, 4, 8, and 16 weeks; or stored at room temperature, 4°C with and without silica gel, or -30°C for 8 and 16 weeks. Podoplanin was assessed using two different antibodies (D2-40, NZ-1) and LYVE-1 was assessed with a single Lyve-1 antibody. The intensity of immunostaining (low, moderate, intense) was compared to control slides that were analyzed the same day they were sectioned.
Summary of Findings:
Podoplanin immunostaining using the D2-40 antibody remained stable in slide-mounted FFPE sections stored at room temperature for 2 weeks or less for scalp skin, hair follicle, and lymph node specimens; however, declines in staining intensity were observed in scalp skin specimens beginning after 4 weeks and hair follicles and lymph node specimens beginning after 8 weeks. In contrast, FFPE slides of skin scalp stored at 4°C with or without silica gel or at -30°C demonstrated comparable D2-40 staining to immediately-stained controls after 8 weeks of storage and only moderate declines in intensity after 16 weeks. Storage of FFPE slides for up to 16 weeks at room temperature did not affect immunostaining with the NZ-1 or Lyve-1 antibodies in scalp skin specimens.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Autopsy
Platform:
Analyte Technology Platform Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature Room temperature
4°C
-30°C
Storage Storage duration 0 weeks
2 weeks
4 weeks
8 weeks
16 weeks