Zinc-Based Fixative Improves Preservation of Genomic DNA and Proteins in Histoprocessing of Human Tissues

Abstract

SUMMARY:Advantageous preservation of histology and detailed cellular morphology has rendered neutral buffered formalin (NBF) the most widely used fixative in clinical pathology. Despite excellent morphology for routine diagnostics, a major drawback of NBF fixation is its detrimental effect on DNA and RNA quality. In addition to complicating analysis of genes and transcripts in complex tissues, NBF denatures proteins and thereby hampers immunohistochemical visualization of certain antigens. In the present study, we evaluated a zinc-based fixative (ZBF) regarding its effects on tissue morphology, quality of genomic DNA, and preservation of protein immunoreactivity in a broad spectrum of tissues. Four different modes of fixation were analyzed: ZBF-paraffin embedding, NBF-paraffin embedding, ZBF-fixation prior to snap-freezing, and immediate snap-freezing. Laserassisted microdissection, allowing retrieval of a defined number of cells for PCR, was used to study DNA quality. Genomic DNA was analyzed using primers for ␤2-microglobulin and the transferrin receptor. Immunohistochemistry was performed using nine antibodies. Tissue microarray blocks were used for analysis of morphology and immunoreactivity. Only slight impairment of morphologic qualities was found after ZBF-paraffin embedding, whereas ZBF prior to freezing resulted in a more crisp morphology compared with routine cryosections. A significantly higher DNA yield was observed in samples isolated from ZBF-paraffin-embedded tissues compared with NBF-paraffin-embedded tissues. Both yield and quality of DNA was comparable in frozen tissues irrespective to prior ZBF fixation. Immunoreactivity in paraffin-embedded tissue was superior in ZBF-fixated tissue compared with NBF-fixated for a majority of tested antibodies. Furthermore, for seven out of nine antibodies, antigen retrieval pretreatment proved unnecessary in ZBF-fixated tissue. Thus, despite a slight impairment of morphology, ZBF preserves protein structures well. We conclude that ZBF is superior to NBF for analysis of DNA and protein expression. Fixation of tissues in ZBF may also be an alternative strategy to freeze storage of tissue specimens, eg, in future bio-banks. (Lab Invest 2003, 83:889 -899).

Gelatin In Situ Zymography on Fixed, Paraffin-embedded Tissue: Zinc and Ethanol Fixation Preserve Enzyme Activity

“…For immunohistochemical analyses, ethanol-and ZBFfixed tissue is found to be advantageous, often eliminating the need for antigen-retrieving procedures commonly performed on NBF-fixed tissue (Beckstead 1994;Ismail et al 2003;Hicks et al 2006). It has also been found that nucleic acids are better preserved in ethanol-and ZBF-fixed material than in NBF-fixed material (Gillespie et al 2002;Leiva et al 2003;Wester et al 2003;Lykidis et al 2007). In the present study, we have shown that paraffin-embedded, ethanol-or ZBF-fixed tissue is also well suited for in situ zymography.…”

“…Fixation results in a reduced volume of the tissue and hence in more-condensed structures (Wester et al 2003), which could possibly lead to more-intense signals when analyzed by in situ zymography. However, the large difference in signal intensity between fixed and frozen tissue can hardly be explained by shrinkage of the tissue alone.…”

“…The exact mode of action for this zinc-based fixative (ZBF) is not fully understood, but it preserves the tissue very well. Judged by light microscopy, the morphology of ZBF-fixed tissue is as good as the morphology of tissue fixed in neutral-buffered formalin (NBF) (Beckstead 1994;Wester et al 2003). In addition, ZBF-fixed tissue is better suited for many histochemical and biomolecular analyses than is NBF-fixed tissue (Beckstead 1994;Ismail et al 2003;Wester et al 2003).…”

The role of endogenous H2S formation in reversible remodeling of lung tissue during hibernation in the Syrian hamster

“…For immunohistological evaluation, lungs were immersion-fixed in a zinc buffer containing 0.5% zinc chloride, 0.5% zinc acetate in 0.1moll -1 Tris base buffer containing 0.05% calcium acetate, pH7.4 (Wester et al, 2003), without inflation or flushing, which leads to preservation of the luminal content. To ensure proper penetration of the fixative, lungs were sliced midway and fixed for 24h at room temperature.…”

Biobanking of fresh frozen tissue: RNA is stable in nonfixed surgical specimens

“…[8][9][10][11][12][13][14] However, studies are scarce concerning the handling of nonfixed tissue between surgery and subsequent freezing and storage at pathology departments. 15 Immediate freezing of tissue specimens after surgery and further storage at -801C generally serve as the standard procedure for the procurement of surgical specimens.…”