The paraffin sections were dewaxed in a xylene solution (10023418, Sinopharm Chemical Reagent Co. Ltd, Beijing, China) and then soaked in Masson’s pine solution (G1006, Servicebio, Wuhan, China) overnight. The sections were incubated in the dye solution composed of an equal ration of Masson’s B solution and Masson’s C solution for 1 min, and then differentiated with 1% hydrochloric acid alcohol. The slices were soaked in Masson’s pine D solution and Masson’s pine E solution for 6 min and 1 min, respectively. After immersion in different solutions, all samples were rinsed with running water. The samples were dried and directly immersed in Masson’s F solution for 30 s. The sections were then rinsed with 1% glacial acetic acid for differentiation and dehydrated with anhydrous ethanol 3 times. Xylene was used to clear the sections for 5 min, and the sections were sealed with neutral gum (10004160, Sinopharm Chemical Reagent Co. Ltd, Beijing, China), observed and images were captured under an optical microscope (NIKON ECLIPSE E100, Nikon, Tokyo, Japan). We selected the same position of the ventricle in each slice from the sample to capture images. Under the microscope, the nucleus and collagen fibres were stained blue. We used ImageJ
25 (link)
software to analyse the images and quantitatively calculate the area percentage of collagen fibres.