Masson s trichrome staining

Heart tissues were fixed at room temperature in buffered formaldehyde solution (10% in PBS) (Sigma, St. Louis, MO, USA) for 24 h, dehydrated using a graded series of ethanol solutions (Sigma, St. Louis, MO, USA), embedded in Paraplast (Sherwood Medical, Mahwah, NJ, USA), and cut into 7-micrometer-thick sections with a pfm rotary 3004 microtome (Leica Microsystems SpA, Milan, Italy) [34 (link)]. Sections were deparaffinized with xylene (Bio-Optica, Milano, Italy) and stained with hematoxylin and eosin (Bio-Optica, Milano, Italy) [35 (link)], Furthermore, Masson’s trichrome staining was performed for the detection of collagen according to the manufacturer’s protocol (Bio-Optica, Milan, Italy), as reported previously [36 (link)]. Sections were evaluated using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy) equipped with a motorized stage and associated with the Leica LAS X Navigator software (Leica Microsystems SpA, Milan, Italy) [37 (link)]. Histologic score was evaluated as described above [38 (link)]. Based on the score value, it is interpreted as:

Negative score (score 0 or 1), the absence of myocardial damage.

Positive score (score 2–7), the existence of damage-

Mild myocardial damage (score 2–3),

-

Moderate myocardial damage (score 4–5),

-

Extensive myocardial damage (score 6–7).

The sections were stained with Masson’s Trichrome staining by following the protocol of Masson’s Trichrome staining kit (Bio-Optica, Milano, Italy). The Zeiss Axiophot microscope (Carl Zeiss AG, Werk Göttingen, Germany) was used to observe illumination through a bright light, and photographs were acquired by a Discovery C30 camera (Tucsen Imaging Technology Co., Ltd. Fujian, China).

Seven-µm thickness sections were stained with Masson’s trichrome staining (Bio-Optica, Milan, Italy), specific for collagen which results blue coloured. The stained sections were examined by means of a Zeiss Axio Imager.A2 microscope (Zeiss, Oberkochen, Germany). The images were acquired by using the Axiocam 503 combined color digital camera (Zeiss, Oberkochen, Germany).