Application suite advanced fluorescence software

The cardiomyocytes were treated with either vehicle alone (control) or vehicle plus GSTM2C constructs at a final concentration of 15 μM and incubated for 2 h. The cells were then loaded with the fluo-4 AM Ca²⁺ indicator and imaged using a Leica SP5 confocal microscope in line scan (x-t) mode. Fluo 4 AM was excited using the 488 nm Argon laser and the line scan was positioned parallel to the longitudinal axis of the cell. Cells were paced at 1 Hz using a 2 ms pulse with a voltage approximately 20% above the contraction threshold. Leica Application Suite Advanced Fluorescence software was used for data acquisition; to correct line scans for background fluorescence and to calculate relative fluorescence (ΔF/F0). Axograph X software (Axograph, Berkely, USA) was used to obtain values for the peak amplitude, rise time from 10% to 90% of the peak and decay time from the peak to 50% of the peak of the ΔF/F0 transients.

For immunostainings, a mixed population of CMPCs and NRVMs was made. Briefly, 7.0 × 10³ CMPCs were seeded onto fibronectin coated coverslips. The next day, 1.5 × 10⁵ NRVM were added and co-cultures were cultured the same as the MEA co-cultures. Cultures were fixed in 4% PFA, before permeabilizing (0.1% Triton) and blocking (1% horse serum/PBS) steps were performed. Cultures were stained with primary antibodies [rabbit anti Connexin 43 (Invitrogen 574366A; 1:200), goat anti cTnT (Hytest LD 16/06-4T19/2; 1:1000X), and mouse anti-beta integrin (Santa Cruz, SC-53711; 1:1,000)] overnight at 4°C before incubation with secondary antibodies (Alexa Fluor-647 donkey anti-mouse IgG (Life Technologies, A31571; 1:250), Alexa Fluor-488 donkey anti-rabbit (Life Technologies, A21206, 1:250) and Alexa Fluor-568 donkey anti goat IgG (Life Technologies, A11057; 1:250), for 2 h in 1% horse serum/PBS. Nuclei were stained with DAPI (Sigma-Aldrich, D9542, 1:40,000). Examination was performed using Leica SPE confocal laser scanning and Leica Application Suite Advanced Fluorescence software.

Cell proliferation was assessed in 3D and 2D cultured cells. Non-trypan blue-stained viable cells were quantified in trypsin-treated spheroids, as well as Ki67-positive cells were determined in 4% paraformaldehyde-fixed spheroid sections (5 µ) by immunocytochemistry as described previously^{18 (link)}. Immunofluorescence analysis was performed using Olympus BX61 microscope. Fluorescence quantification was performed using Leica Application Suite Advanced Fluorescence software and ImageJ software. Cell proliferation rate was also measured by bromodeoxyuridine (BrdU) incorporation in 2D cultured cells using the recommendation included in the commercial assay (11647229001, Roche Diagnostics, Basel, Switzerland)^{18 (link)}. Data are shown as the relative absorbance at 370 nm, using as reference wavelength 492 nm (Absorbance, %, fold over control) using an Infinite 200 PRO Microplate Reader (TECAN, Männedorf, Switzerland).

Images of DAB-stained sections were taken using a Zeiss Axio Imager M1 with charge-coupled device camera using a 20× objective. Immunofluorescent sections were imaged using 40×, 1.15 numerical aperture and 63×, 1.30 numerical aperture oil objectives on a Leica TCS SPE confocal microscope using Leica Application Suite Advanced Fluorescence software. Z-stack images of fluorescent lung sections were acquired by sequential scanning every 1 µm and taken at a 2,048 × 2,048 pixel size, with a line average of 2 to reduce noise. Stacks were reconstructed using FIJI (NIH), and Photoshop (Adobe) software was used to change brightness and contrast of the images.
To quantify double labeling, we used an unbiased approach. For example, to examine whether GFP expression in GFAPcre-GFP mice was present in GFAP + cells, lung sections from three animals were immunostained for GFAP with a red (Alexafluor 555) secondary. When a GFP-expressing cell was identified in the green channel, the channel was switched to red to determine whether it also stained for GFAP. Conversely, to evaluate which percent of GFAP immunostained cells express GFP, GFAP + cells were identified in the red channel then the channel switched to green to score GFP expression. At least 100 cells from each mouse were counted for each of these questions.