Type 1collagen

Samples of cardiac tissue, from both noninfected and T. cruzi-infected mice, were frozen in Tissue-Tek O.C.T Compound (Sakura, Zoeterwoude, The Netherlands) and stored in liquid nitrogen. Heart cryosections were fixed in cold acetone, air-dried and stored at -20 °C. Sections were washed in PBS and then blocked with PBS containing 4% serum albumin bovine (BSA; PBS + BSA) before immunostaining. The samples were incubated overnight at 4 °C with anti-fibronectin (1:600, Sigma Chemical Co., St Louis - USA) or type 1 Collagen (1:400; Life Technologies Co., Carlsbad, CA - USA) antibodies followed by washing with PBS + BSA. The antigen-antibody complex was detected by incubation for 1 h at 37 °C with TRITC-conjugated secondary antibody (1:400, Sigma). After washing, the sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI), a DNA staining, and mounted with the anti-fading DABCO® (Sigma). Images were acquired under a Zeiss Axio Imager M2 fluorescence microscope using AxioVision (Zeiss) digital image processing software.
For the type I collagen bioimage analysis, at least five different fields were captured from each group (uninfected and infected), and fluorescence images were segmented and the mean intensity measured using Knime workflow.³³ (link)