The Application of Guanidinium to Improve Biomolecule Quality in Fixed, Paraffin-embedded Tissue

Abstract

Neutral buffered formalin (NBF) is the most common fixative in clinical applications. However, NBF damages proteins and nucleic acids, limiting the quality of proteomic and nucleic acid-based assays. Prior studies have demonstrated that BE70, a fixative of buffered 70% ethanol, has many benefits over NBF but the degradation of proteins and nucleic acids in archival paraffin blocks remain a challenge. Thus, we evaluated the addition of guanidinium salts to BE70 with the hypothesis that this may protect RNA and protein. Guanidinium salt supplemented BE70 (BE70G)-fixed tissue is comparable with that of BE70 via histology and immunohistochemistry. Western blot analysis also revealed that HSP70, AKT, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression signals in BE70G-fixed tissue were higher than those in BE70-fixed tissue. The quality of nucleic acids extracted from BE70G-fixed, paraffin-embedded tissue was also superior, and BE70G provides improved protein and RNA quality at shorter fixation times than its predecessors. The degradation of proteins, AKT and GAPDH, in archival tissue blocks is also decreased with the addition of guanidinium salt to BE70. In conclusion, BE70G fixative improves the quality of molecular analysis with more rapid fixation of tissue and enhanced long-term storage of paraffin blocks at room temperature for evaluation of protein epitopes.

Keywords: RNA; alcohol; fixation; formaldehyde; guanidinium; histology; immunohistochemistry; protein.

Figure 1.

Quantity and quality of protein extracted from NBF-, BE70-, or BE70G-fixed tissues. (A) Protein extraction yield from each condition. The protein was extracted from mouse kidney and liver in NBF-, BE70-, or BE70G-fixed and paraffin-embedded tissue cores. The bar graph shows the relative means of protein extraction yields. (B) Protein integrities of different fixatives-fixed tissues were assessed by Western blotting. Protein extracted from each condition was probed with antiserine/threonine protein kinase (AKT) and GAPDH antibodies. (C) Relative expression signal of AKT and GAPDH expression signal under each condition is presented as a bar graph. The relative protein signal of each entity was normalized to 1-day BE70G-fixed tissue (1.00). Quantitative analyses were performed using ImageQuant version 5.2. Data are presented as the mean ± standard deviation (SD) from three independent experiments. Abbreviations: NBF, neutral buffered formalin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. *p<0.05, **p<0.01, ***p<0.001.

Figure 2.

Protein quantity and quality according to the fixation time in NBF, BE70, and BE70G. (A) The protein was extracted from mouse liver in NBF-, BE70-, or BE70G-fixed and paraffin-embedded tissue cores. The bar graph shows the extraction yields. (B) Relative serine/threonine protein kinase (AKT) expression signal under each condition. The quality of proteins in different fixatives according to fixation time was assessed by Western blotting. Quantitative analyses were performed using ImageQuant version 5.2. Protein quantity and quality according to the fixation time in a single fixative are presented as miniature graphs in the right panel. The relative protein extraction yield and expression signal of each entity was normalized to 1-day BE70G-fixed tissue (1.00). Data are presented as the mean ± standard deviation (SD) from three independent experiments. Abbreviations: NBF, neutral buffered formalin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. *p<0.05, **p<0.01, ***p<0.001.

Figure 3.

The quality of protein immunoreactivity in NBF-, BE70-, or BE70G-fixed tissue sections under different storage conditions. The protein immunoreactivity in different fixatives according to fixation time was assessed by Western blotting. (A) Relative AKT expression signal according to the fixation time in NBF, BE70, and BE70G. (B) Relative GAPDH expression signal according to the fixation time in NBF, BE70, and BE70G. Protein quantity and quality according to the fixation time in a single fixative are presented as miniature graphs in the right panel. The relative protein extraction yield and expression signal of each entity were normalized to 1-day BE70G-fixed tissue (1.00). Data are presented as the mean ± standard deviation (SD) from three independent experiments. Control, BE70G (0 h); Abbreviations: NBF, neutral buffered formalin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; VD, vacuum + drierite; HC, humidity chamber. *p<0.05, **p<0.01, ***p<0.001.

Figure 4.

RNA quantity and quality according to the fixation time in NBF, BE70, and BE70G. (A) RNA extraction yield under each condition. The RNA was extracted from mouse liver in NBF-, BE70-, or BE70G-fixed and paraffin-embedded tissue cores. The relative RNA extraction yield of each entity was normalized to 1-day BE70G-fixed tissue (1.00). (B) RNA integrity derived from each fixative condition is presented as the mean cycle threshold (Ct) value of the beta-Actin (ACTB). Gene expression levels are shown as box plots. Relative RNA extraction yield and Ct values according to fixation time in a single fixative are presented as miniature graphs in the right panel. Data are the mean ± SD from three independent experiments. Abbreviations: NBF, neutral buffered formalin; ACTB, beta-Actin. *p<0.05, **p<0.01, ***p<0.001.

Figure 5.

DNA quantity and quality according to the fixatives. (A) The DNA was extracted from mouse kidney and liver in NBF-, BE70-, or BE70G-fixed and paraffin-embedded tissue cores. (B) Amplification of DNA derived from each condition was assessed using the BioScore Screening and amplification kit. Data are the mean ± SD from three independent experiments. Abbreviation: NBF, neutral buffered formalin. *p<0.05, **p<0.01, ***p<0.001.

Figure 6.

Comparison between BE70G, BE70, and NBF fixation with representative mouse tissues obtained from stomach (A), kidney (B), brain (C), and heart (D). The overall histomorphological features of BE70G are similar to those of NBF. But kidney tissue fixed in BE70G shows blurred nuclear chromatin details and perinuclear halo due to nuclear shrinkage, which is observed in especially renal tubular cells (inlet of B). Scale bar, 100 µm. Abbreviation: NBF, neutral buffered formalin.

Figure 7.

Immunohistochemical stainings for cytokeratin 14 with mouse ear (A), Ki-67 with mouse spleen (B), and E-cadherin with mouse small intestine (C). The cytoplasmic, nuclear, and membranous staining patterns of BE70G are definite and show similar intensity of BE70 or NBF. Scale bar, 100 µm. Abbreviation: NBF, neutral buffered formalin.