Immunohistological Analysis of Cardiac Fibrosis

Paraffin-embedded hearts (vehicle n = 6, empa n = 8) were cut 4–6 μm thick and stained. Immunohistological techniques were performed as previously described^{17 (link)}. Heart sections were incubated with primary antibodies against rat macrophages (ED1; #MCA341R, Bio-Rad), fibronectin (FN; #ab23751, abcam), and collagen I (Col I; #131001, SouthernBiotech). Vectashield mounting medium with DAPI (#H-1200, Vector Laboratories) was used to stain nuclei. Semi-quantitative analyses of immunohistochemistry and histology were conducted without knowledge of the specific treatment and performed using Pannoramic MIDI II slide scanner (3D Histech, Hungary), CaseViewer and Image J software. For the histological and cardiomyocyte analysis, 5 hearts of each group were examined. Picking out cardiac areas for immunohistology were standardized and uniformly distributed over the entire cross-section. Ten to fifteen different areas of each heart were analyzed. For the determination of the perivascular fibrosis, all vessels in the left ventricle were evaluated.

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Kräker K., Herse F., Golic M., Reichhart N., Crespo-Garcia S., Strauß O., Grune J., Kintscher U., Ebrahim M., Bader M., Alenina N., Heuser A., Luft F.C., Müller D.N., Dechend R, & Haase N. (2020). Effects of empagliflozin and target-organ damage in a novel rodent model of heart failure induced by combined hypertension and diabetes. Scientific Reports, 10, 14061.

Publication 2020

Col I Vectashield mounting medium with DAPI Pannoramic MIDI II slide scanner

Antibodies Cardiomyocyte Collagen i Cross over Dapi Fibronectin Fibrosis Heart Immunohistochemistry Left ventricle Macrophages Nuclei Paraffin Stain Vector Vessels

Corresponding Organization :

Other organizations : Charité - Universitätsmedizin Berlin, German Centre for Cardiovascular Research, Technische Universität Berlin, Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Max Delbrück Center

Top 5 similar protocols

Variable analysis

independent variables

Treatment (vehicle vs. empagliflozin)

dependent variables

Macrophage infiltration (ED1 immunohistochemistry)
Fibronectin expression (fibronectin immunohistochemistry)
Collagen I expression (collagen I immunohistochemistry)
Perivascular fibrosis (histological analysis)

control variables

Heart section thickness (4-6 μm)
Staining techniques (previously described in reference 17)
Image analysis software (Pannoramic MIDI II, CaseViewer, ImageJ)
Sample size (vehicle n = 6, empagliflozin n = 8)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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