Safefix solution

Manufactured by Thermo Fisher Scientific

SafeFix solution is a laboratory product designed to safely and effectively fix biological samples for various analyses. It is a ready-to-use, stabilizing fixative solution that preserves the structural integrity and morphology of cells, tissues, or other biological materials. The core function of SafeFix solution is to enable consistent sample preparation for downstream applications such as microscopy, flow cytometry, and immunohistochemistry.

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SafeFix (5 citations)

6 protocols using safefix solution

Histopathological Scoring of Cardiac Inflammation and Fibrosis

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Heart tissue was fixed in SafeFix solution (Thermo Fisher Scientific), embedded, and cut into 5-µm serial sections. Ventricular inflammation was scored by microscopic assessment of the area of infiltration with hematopoietic cells on H&E-stained sections according to the following scale: grade 0, no inflammation; grade 1, <10% of the heart section is infiltrated; grade 2, 10–30%; grade 3, 30–50%; grade 4, 50–90%; and grade 5, >90%. Ventricular fibrosis was scored on Masson’s trichrome–stained sections according to the same scale. Atrial inflammation and fibrosis were scored on the following scale: 0, none; 1, mild; 2, moderate; and 3, severe. Grading was performed by two independent, blinded investigators and averaged (Ciháková et al., 2004 (link)).

Diny N.L., Baldeviano G.C., Talor M.V., Barin J.G., Ong S., Bedja D., Hays A.G., Gilotra N.A., Coppens I., Rose N.R, & Čiháková D. (2017). Eosinophil-derived IL-4 drives progression of myocarditis to inflammatory dilated cardiomyopathy. The Journal of Experimental Medicine, 214(4), 943-957.

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Myocarditis and Cardiac Fibrosis Assessment

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Mice were evaluated for the development of EAM and DCMi on days 21 and 63, respectively. Heart tissues were fixed in SafeFix solution (Thermo Fisher Scientific). Tissues were embedded longitudinally, and 5-µm serial sections were cut and stained with H&E or Masson’s trichrome blue (HistoServ). Myocarditis severity was evaluated by H&E staining of myocardium area infiltrated with hematopoietic cells, according to the following scoring system: grade 0, no inflammation; grade 1, <10% of the heart section is involved; grade 2, 10–25%; grade 3, 25–50%; grade 4, 50–75%; grade 5, >75%. Grading was performed by grading five sections per heart by two independent, blinded investigators and averaged. Cardiac fibrosis was evaluated by measuring the area of blue staining of fibrosis as a proportion of heart cross section.

Wu L., Ong S., Talor M.V., Barin J.G., Baldeviano G.C., Kass D.A., Bedja D., Zhang H., Sheikh A., Margolick J.B., Iwakura Y., Rose N.R, & Čiháková D. (2014). Cardiac fibroblasts mediate IL-17A–driven inflammatory dilated cardiomyopathy. The Journal of Experimental Medicine, 211(7), 1449-1464.

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Histological Evaluation of Myocarditis Severity

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Myocarditis severity was evaluated by histology on days 21. Heart tissues were fixed in SafeFix solution (Thermo Fisher Scientific), embedded and cut into 5 μm serial sections. Sections were stained with H&E and ventricular inflammation was scored via a microscope by two independent blinded investigators and averaged using the following criteria for hematopoietic infiltrates: grade 0, no inflammation; grade 1, < 10% of the heart section is involved; grade 2, 10%–30%; grade 3, 30%–50%; grade 4, 50%–90%; grade 5, > 90% (Ciháková et al., 2004 (link)).

Hou X., Chen G., Bracamonte-Baran W., Choi H.S., Diny N.L., Sung J., Hughes D., Won T., Wood M.K., Talor M.V., Hackam D.J., Klingel K., Davogustto G., Taegtmeyer H., Coppens I., Barin J.G, & Čiháková D. (2019). The Cardiac Microenvironment Instructs Divergent Monocyte Fates and Functions in Myocarditis. Cell reports, 28(1), 172-189.e7.

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Determination of Infarct Size in Mouse Hearts

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Heart tissues were fixed in SafeFix solution (Thermo-Fisher Scientific), embedded in paraffin, and cut into 5-μm-thick sections. Hematoxylin and eosin (H&E) and Masson’s trichrome staining (HistoServ) were performed on paraffin-embedded sections to determine the infarct size. Three sections were used to calculate the infarct size, i.e. a section right below the ligation site (Section A), a section near the apex (Section C) and Section B at a midpoint of sections A and C. The infarct size was calculated as the total infarct circumference of all three sections divided by the total left ventricle circumference of all three sections [77 ].

Chen G., Bracamonte-Baran W., Diny N.L., Hou X., Talor M.V., Fu K., Liu Y., Davogustto G., Vasquez H., Taegtmeyer H., Frazier O.H., Waisman A., Conway S.J., Wan F, & Čiháková D. (2018). Sca-1+ cardiac fibroblasts promote development of heart failure. European journal of immunology, 48(9), 1522-1538.

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Histological Evaluation of Myocarditis Severity

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Histological Analysis of Cardiac Inflammation

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Heart tissue from immunized mice was fixed in SafeFix solution (Thermo Fisher Scientific). Serial sections (5 μm) were cut, embedded, and stained with hematoxylin and eosin (H&E) to analyze the heart morphology. Histological grades were scored by microscopic assessment of the area of hematopoietic cell infiltration on H&E stained sections. Inflammation of heart tissue was scored according to the following system: grade 0, no inflammation; grade 1, < 10% of the heart section is inflamed; grade 2, 10-25%; grade 3, 25-50%; grade 4, 50-75%; grade 5, > 75%. Grading was performed by two independent, blinded investigators who assessed five areas per heart, after which grades were averaged. Heart samples were also stained with Masson's trichrome to analyze myocardial fibrosis and detect collagen fiber density. The fraction stained with aniline blue was calculated as the blue-stained area divided by the total field. For measurements of cardiomyocyte area, the heart cross-sections were stained with FITC-labeled wheat germ agglutinin (WGA; Sigma-Aldrich Corp.) to visualize the myocyte membranes.

Wu J., Sun P., Chen Q., Sun Y., Shi M., Mang G., Yu S., Zheng Y., Li Z., Sun M., Fang S., Zhang Y., Tian J., Mingyan E., Zhang M, & Yu B. (2019). Metabolic reprogramming orchestrates CD4⁺ T-cell immunological status and restores cardiac dysfunction in autoimmune induced-dilated cardiomyopathy mice. Journal of molecular and cellular cardiology, 135.

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