Factors influencing the degradation of archival formalin-fixed paraffin-embedded tissue sections

Abstract

The loss of antigenicity in archival formalin-fixed paraffin-embedded (FFPE) tissue sections negatively affects both diagnostic histopathology and advanced molecular studies. The mechanisms underlying antigenicity loss in FFPE tissues remain unclear. The authors hypothesize that water is a crucial contributor to protein degradation and decrement of immunoreactivity in FFPE tissues. To test their hypothesis, they examined fixation time, processing time, and humidity of storage environment on protein integrity and antigenicity by immunohistochemistry, Western blotting, and protein extraction. This study revealed that inadequate tissue processing, resulting in retention of endogenous water in tissue sections, results in antigen degradation. Exposure to high humidity during storage results in significant protein degradation and reduced immunoreactivity, and the effects of storage humidity are temperature dependent. Slides stored under vacuum with desiccant do not protect against the effects of residual water from inadequate tissue processing. These results support that the presence of water, both endogenously and exogenously, plays a central role in antigenicity loss. Optimal tissue processing is essential. The parameters of optimal storage of unstained slides remain to be defined, as they are directly affected by preanalytic variables. Nevertheless, minimization of exposure to water is required for antigen preservation in FFPE tissue sections.

Figure 1.

Immunohistochemical analysis of AQP1 expression according to fixation time and tissue processing time. We stained tissue sections of mouse kidney formalin-fixed paraffin-embedded (FFPE) tissue after a 12- or 24-hr fixation period, as well as tested 4-hr or 8-hr tissue processing time after a 0-week (a) and 2-week (b) storage room temperature. All photomicrographs ×250.

Figure 2.

Immunohistochemical analysis of AQP1 expression in formalin-fixed paraffin-embedded (FFPE) tissue sections of mouse kidney under different storage conditions for 3 months. (a) RT-Vac + Drierite, (b) RT-HC, (c) 4C-Vac + Drierite, (d) 4C–HC, (e) 30C-Vac + Drierite, (f) 30C–HC, (g) 37C-Vac + Drierite, (h) 37C–HC. All photomicrographs ×200. HC, humidity chamber; RT, room temperature; Vac, vacuum packed.

Figure 3.

Assessments of protein profiles according to storage conditions. Protein quality was analyzed on the Agilent 2100 bioanalyzer, using 1 µg of denatured total protein extracted from mouse kidney formalin-fixed paraffin-embedded tissue sections under different storage conditions. Representative data are presented as an eletropherogram, in which the first peak on the left is a protein marker.

Figure 4.

Assessment of protein immunoreactivity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by Western blotting. (a) Western Blot by anti-GAPDH antibody. Lane 1, RT-Vac + Drierite; lane 2, RT-HC; lane 3, 4C-Vac + Drierite; lane 4, 4C–HC; lane 5, 30C-Vac + Drierite; lane 6, 30C–HC; lane 7, 37C-Vac + Drierite; lane 8, 37C–HC. (b) Quantitative analysis of Western blot (ImageQuant Program version 5.2, Piscataway, NJ). HC, humidity chamber; RT, room temperature; Vac, vacuum packed.

Figure 5.

Assessment of protein immunoreactivity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in formalin-fixed, paraffin-embedded tissue sections under different storage conditions for 3 months by a novel protein array platform. The expressional values are represented as box graphs as follows: vacuum + Drierite (a), humidity chamber (b), and merged (c). Values shown are mean ± SE.