Immunofluorescence and assessment of ITGA5 expression, macrophage, and myofibroblast density

Immunofluorescence was used to study the time course of ITGA5 localization in infarct macrophages and to assess the effects of myeloid and macrophage-specific ITGA5 loss on macrophage and myofibroblast density and on the number of mature coated vessels In order to study the time course, and cellular localization of ITGA5 activation in infarct macrophages, ITGA5 (Abcam, ab150361), and anti-GFP (Abcam, ab6662), or Wheat Germ Agglutinin, Alexa Fluor™ 633 (WGA AF633, Thermo Fisher Scientific, Waltham, MA) staining was performed on sections from infarcted CSF1R-EGFP hearts at baseline, and after 24 h, 3 days, 7 days and 28 days of coronary occlusion (control: n = 6; 24 h: n = 6; 3 days, n = 5; 7 days, n = 6; 28 days, n = 6). Sections were deparaffinized in Xylene, rehydrated in consecutive graded washes of ethanol, and exposed to heat-mediated antigen retrieval for 30 min with citrate buffer, pH 6.0. Sections were washed three times with TBS (5 min each) and then permeabilized with 0.1% Triton (Sigma-Aldrich, T8787) in TBS (T-TBS) for 10 min, followed up with non-specific protein blocking via incubation for 1 h in 10% donkey serum (Sigma Aldrich, D9663) in TBS. Slides were then incubated overnight at 4 °C with anti-ITGA5 and anti-GFP primary antibodies. Sections were then washed with TBS and incubated with fluorescently labeled secondary antibodies for 1 h at room temperature. Antibodies dilutions were prepared in TBS. Autofluorescence quenching was performed using TrueBlack Lipofuscin Autofluorescence Quencher (Biotium, #3007) and mounted using Fluoro-Gel II with DAPI (EMS, 50-246-93). Fluorescently stained sections were scanned using Zen 2.6 Pro software and the Zeiss Imager M2 microscope (Carl Zeiss, White Plains, NY). In addition, Leica SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) was employed for the acquisition of high-resolution images. The microscope is housed in a controlled environment to minimize vibrations in the Analytical Imaging facility of Albert Einstein College of Medicine. Leica LAS X software was utilized to control the microscope settings and for image acquisition. Laser power and gain settings were optimized to ensure minimal photobleaching, while maintaining adequate signal-to-noise ratios. Quantitative analysis was performed by counting the number of ITGA5-positive and GFP-positive macrophages in 15 fields from 2 different sections from each animal. To assess the number of macrophages in the infarcted and remodeling myocardium, sections were stained with Mac-2 antibody (Cedarlane, CL8942AP) in the infarcted ITGA5 fl/fl, Myα5KO and iMaα5KO hearts (Mac-2 staining: 7 days group: ITGA5 fl/fl n = 19, Myα5KO n = 12, iMaα5KO n = 5; 28-day group: ITGA5 fl/fl n = 22, Myα5KO n = 13, iMaα5KO n = 9). Macrophage quantitation was performed, using an AI model (Zen 2.6 Pro software) in which macrophages were identified as the DAPI-positive nuclei surrounded by Mac2+ profiles. In order to assess myofibroblast density, we performed immunofluorescent staining with an anti-α-SMA antibody (Sigma, F3777). Myofibroblasts were identified as spindle-shaped α-SMA-positive cells located outside the vascular media. An AI-based model (Zen 2.6 Pro software) was trained on multiple fields of different regions of the myocardium to identify myofibroblasts. Objects of interest were defined as the DAPI-positive nuclei surrounded by α-SMA profiles, excluding vascular smooth muscle cells. Quantitative analysis was performed using 10 fields from 2 different sections from each animal (α-SMA staining: 7 days group: ITGA5 fl/fl n = 19, Myα5KO n = 12, iMaα5KO n = 5; 28-day group: ITGA5 fl/fl n = 22, Myα5KO n = 13, iMaα5KO n = 9). Negative controls were performed to confirm the specificity of the antibodies using sections in which the primary antibody was substituted by species-specific IgG at the same concentration. Quantitative histological studies were performed by an investigator (R.L.) blinded to the study groups. Cell density was expressed as cells/mm².