Anti rabbit horseradish peroxidase hrp reagent

Four weeks after the AB operation, the mouse heart was removed, fixed with 10% paraformaldehyde, embedded in paraffin, and sectioned (5 μM thickness). Hematoxylin and eosin (HE) staining was used to evaluate the area of cardiomyocytes. The collagen deposition fraction was evaluated by picric acid red (PSR) staining. The myocardial cell area (at least 200 per group) and left ventricular collagen fraction were calculated by a quantitative digital image analysis system (Image-Pro Plus, IPP, version 6.0). Heart sections were incubated with anti-CD45 (Abcam, 1 : 100 dilution) or anti-CD68 (Abcam, 1 : 100 dilution) antibodies and then crosslinked with anti-rabbit horseradish peroxidase (HRP) reagent (Gene-tech, Shanghai, China). Finally, the DAB substrate kit (Gene-Tech, Shanghai, China) was used for coloration. A fluorescence microscope was utilized to acquire images and count CD45⁺CD68⁺ cells (10 fields for each heart).

Triphenyltetrazolium chloride (TTC, 1%, Sigma, USA) staining was used to evaluate the MI area and morphological changes in the heart. For the infarct area calculation, Image-Pro Plus 6.0 was used to analyse 6 sections from each heart and 6 hearts from each group. Macrophages were subjected to immunohistochemistry staining for CD68. After dehydration, antigen repair was conducted at a high temperature and pressure, and the sections were sealed with 8% goat serum. The heart sections were incubated with an anti-CD68 antibody (Abcam, 1:100 dilution) and then with an anti-rabbit horseradish peroxidase (HRP) reagent (Gene Tech, Shanghai, China). A peroxide-based substrate DAB kit (Gene Tech, Shanghai, China) was used for coloration. Macrophages were subjected to immunohistochemistry staining for F4/80, NOS2, and CD206 to detect M1 and M2 positive cells. The heart sections were incubated with an anti- F4/80, NOS2, and/or CD206 antibody (Abcam, 1:100 dilution). Secondary antibody goat anti-mouse/rabbit IRdye 800CW (LI-COR) were used. The nuclear was stained with DAPI. We counted the number of F4/80, NOS2, or F4/80, CD206 positive cells for each group (10 field for each heart).