2.7. Immunofluorescence in Cardiac Tissue

Heart tissues were kept and washed in phosphate-buffered saline (PBS). Thereafter, the tissues were soaked in 4% paraformaldehyde for 48 h at 4 °C. After fixation, the samples were transferred to 30% sucrose in PBS at 4 °C overnight. The tissues were embedded in an embedding medium for frozen tissue specimens (catalog number: 4583, Sakura Finetek USA, Inc., Torrance, CA, USA). Blocks were cut with the Cryostat Microm HM 525 instrument (ThermoFisher Scientific, Walldorf, Germany) with a thickness of 10 µm at −25 °C and were placed on a polysine adhesive microscope slide (HDAS002). Sections of the heart were obtained following the protocols of Givvimani and colleagues [49]. The slides were processed and incubated with anti-MMP-2 (catalog number: sc-13595, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or MMP-9 (catalog number: AB19016, Merck, Germany) antibodies or anti-collagen type I (catalog number: ab34710, Abcam) at 4 °C overnight. Following overnight primary antibody application, the slides were washed with 0.1 M PBST 3 times (10 min each) and the secondary antibody (Alexa Fluor^® 488 (catalog number: ab150077, Abcam) or Alexa Fluor^® 594 (catalog number: sc-515884, Santa Cruz Biotechnology, Inc., Dallas, TX, USA)) was applied for 2 h at 37 °C. Next, the slides were washed with PBST and stained with DAPI (catalog number: D9542, Sigma-Aldrich, USA) for 10 min. In the last step, the slides were washed with PBS 3 times (5 min each), then mounted and visualized with fluorescence via a laser scanning confocal microscope (Carl Zeiss, Germany) with an appropriate filter. Confocal imaging is detected by 10X objective lens. Images were acquired with 1024 × 1024x pixels. Fluorescence intensity of DAPI, MMP-2, MMP-9, and collagen type I (average fluorescent intensity per pixel) was measured in each projection by Zeiss Zen Blue analysis wizard software.