Evaluation of non-formalin tissue fixation for molecular profiling studies

Abstract

Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analyzed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. Complete protocols and additional discussion of relevant issues are available on an accompanying website (http://cgap-mf.nih.gov/).

Figure 1.

Comparison of the histological quality of 5-μm-thick sections of prostate tissue stained with H&E. A: Normal prostate. Original magnification, ×100. A: Formalin-fixed, paraffin-embedded. B: 70% ethanol-fixed, paraffin-embedded. C: 70% ethanol-fixed, polyester wax-embedded. D: Frozen. Note the comparable staining quality and architecture for the formalin-fixed and ethanol-fixed tissues, which are superior to the frozen. B: High-grade prostatic intraepithelial neoplasia from ethanol-fixed, paraffin-embedded prostate tissue showing nuclear overlap and fine nuclear detail, including prominence of nucleoli. H&E stained, original magnification, ×200.

Figure 2.

Immunohistochemical stain of normal prostate tissue for PSA. Original magnification, ×100, hematoxylin counterstain. A: Ethanol-fixed, paraffin-embedded. B: Formalin-fixed, paraffin-embedded. Both preparations show comparable staining with positive glandular epithelial cells and negative stroma.

Figure 3.

A: Protein recovery from prostate tissue sections. Samples were either snap frozen (Froz), ethanol-fixed and paraffin-embedded (Et/Para), ethanol-fixed and polyester wax-embedded (Et/PEW), or formalin-fixed and paraffin-embedded (Form/Para). B: Immunoblot for PSA. Approximately 35,000 prostate epithelial cells were microdissected from histological sections using LCM. Two separate microdissections from the ethanol-fixed, paraffin-embedded sample were performed to assess reproducibility.

Figure 4.

Two-dimensional PAGE analysis of ethanol-fixed, paraffin-embedded prostate tissue.

Figure 5.

Layered expression scanning using an ethanol-fixed, paraffin-embedded prostate which has been transferred onto nitrocellulose and stained with Coomassie blue. The labeled regions demonstrate that the protein staining pattern retains characteristic prostate structures.

Figure 6.

A: Denaturing agarose gel of total RNA from prostate tissue sections that were either frozen, ethanol-fixed, polyester wax-embedded (EtOH/PEW), ethanol-fixed, paraffin-embedded (EtOH/Pf), or formalin-fixed, paraffin-embedded (Form/Pf). B: RT-PCR for actin. Approximately 15,000 epithelial cells were microdissected from frozen or ethanol-fixed, paraffin-embedded (EtOH/Pf) prostate sections. Actin cDNA and prostate RNA from cell culture are included as positive controls. The arrow indicates the 220-bp band corresponding to actin.

Figure 7.

A: Analysis of total DNA quality in prostate tissue that was either ethanol-fixed and paraffin-embedded (Et/Para), ethanol-fixed and polyester wax-embedded (Et/PEW), or formalin-fixed and paraffin-embedded (Form/Para). Each sample was loaded onto a 1% agarose gel. DNA was visualized with ethidium bromide staining. B: PCR-based analysis of DNA quality in prostate tissue that was either snap frozen, ethanol-fixed and paraffin-embedded (Et/Para), ethanol-fixed and polyester wax-embedded (Et/PEW), or formalin-fixed and paraffin-embedded (Form/Para). The PCR product from amplification of microsatellite marker D17S926 was electrophoresed on a 6% denaturing acrylamide gel and visualized by autoradiography. All samples were analyzed in duplicate.