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, 58 (4), 2448

Wheat Germ Agglutinin Staining as a Suitable Method for Detection and Quantification of Fibrosis in Cardiac Tissue After Myocardial Infarction

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Wheat Germ Agglutinin Staining as a Suitable Method for Detection and Quantification of Fibrosis in Cardiac Tissue After Myocardial Infarction

B Emde et al. Eur J Histochem.

Abstract

The quantification of fibrotic tissue is an important task in the analysis of cardiac remodeling. The use of established fibrosis staining techniques is limited on frozen cardiac tissue sections due to a reduced color contrast compared to paraffin embedded sections. We therefore used FITC-labeled wheat germ agglutinin (WGA), which marks fibrotic tissue in comparable quality as the established picrosirius red (SR) staining, for the staining of post myocardial infarction scar tissue. The fibrosis amount was quantified in a histogram-based approach using the non-commercial image processing program ImageJ. Our results clearly demonstrate that WGA-FITC is a suitable marker for cardiac fibrosis in frozen tissue sections. In combination with the histogram-based analysis, this new quantification approach is i) easy and fast to perform; ii) suitable for raw frozen tissue sections; and iii) allows the use of additional antibodies in co-immunostaining.

Conflict of interest statement

Conflicts of interest: the authors declare no conflicts of interest.

Figures

Figure 1.
Figure 1.
Comparison of picrosirius red staining of paraffin embedded (A,C) and raw frozen tissue (B, D). Images were taken with 4x objective (A,B) and 20x objective (C,D) of post-MI heart sections where a part of the scar (dark red dye) and the remote myocardium (yellow dye) is visible.
Figure 2.
Figure 2.
Sirius red stain (left image) and WGA-FITC stain (right image) of mouse heart frozen tissue sections, after 45 min ischemia and four weeks reperfusion. All hearts show a scar in the myocardium of the left ventricle in different dimensions.
Figure 3.
Figure 3.
A) Comparison of numerical values of planimetric scar size analyses between wheat germ agglutinin-FITC (WGA, P) and picrosirius red (SR, P) stained heart cryosections. B) Correlation diagram of these data. R2=0.9559, P=0.004.
Figure 4.
Figure 4.
A) WGA (green) and Collagen I (red) co-staining of heart slices of one exemplary I/R-experiment. B) 20x magnification of WGA/Collagen I co-stained heart showing scar tissue and normal tissue. Nuclei are stained with DAPI (blue). C) 40x magnification of a WGA/Collagen I co-stained heart. Nuclei are stained with DAPI (blue). Staining in all panels from left to right: WGA (green, FITC), Collagen I (red, Cy3), Overlay.
Figure 5.
Figure 5.
Histogram-based quantification of fibrosis amount. A,B) Exemplary traces of the background area and the complete heart for WGA-stained (A) and SR-stained (B) section. C) Overlay of the green histograms of the background (black border) and of the complete heart (red border). The Background was extrapolated to the pixel amount of the whole heart for a direct comparison. From brightness level ~70 (black arrow) the pixel of the whole heart over the background represent the fibrotic fraction. D) Red histograms of the heart after SR staining for background (black border) and complete heart (red border) with extrapolated background as described above.
Figure 6.
Figure 6.
A) Comparison of numerical values of planimetric (WGA, P) and histogram-based (WGA, H) analysis of fibrosis amount in all five examined hearts after WGA-FITC staining. B) Correlation diagram of the planimetric and histogram-based automatically assessed data. R2=0.8318, P=0.0309.

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