Hoescht 33258

Calcium assay is performed using labeling protocol as described^{61 (link)}. After 11-day treatment with PPARα agonist and/or PGC1/PPARα target-directed siRNAs, 50 µL of media was removed and replaced in each well by a 2× calcium dye solution consisting in Fluo-4 NW dye (Invitrogen), 1.25 mM Probenecid F-127 (Invitrogen) and 100 µg/mL Hoescht 33258 (diluted in water, ThermoFisher) diluted in warm Tyrode’s solution (Sigma). The plate was placed back in the 37 °C 5% CO₂ incubator for 45 min. After incubation time, cells were washed four times with fresh pre-warmed Tyrode’s solution by removing 50 µL of media and adding 50 µL of Tyrode’s solution using a 16 channel pipette. hiPSC-CMs were then automatically imaged with ImageXpress Micro XLS microscope (Molecular Devices) at an acquisition frequency of 100 Hz for a duration of 5 s with an excitation wavelength of 485/20 nm and emission filter 525/30 nm. A single image of Hoescht was acquired before the time series. Fluorescence over time quantification and trace analysis were automatically quantified using custom software packages developed by Molecular Devices and Colas lab.

Immunohistochemistry experiments on coronal brain sections (50–100 μm, vibratome, Microm; 14 μm, cryostat, Leica) were carried out as described previously (Cloarec et al., 2016 (link)) with the following primary (anti-Iba1: 1/500, Wako; anti-Cd68, Ed1 clone: 1/200, Millipore) and secondary (Alexa Fluor 568 or 647-conjugated goat anti-rabbit or anti-mouse IgGs; Life Technologies) antibodies. Hoescht 33258 (1:2000, Sigma) was used for nuclei staining.
For tissue clearing experiments (see next subsection), whole infected brains were first immunostained as follows. Tissue samples were dehydrated in methanol/1X PBS series: 20, 40, 60, 80, 100 × 2 for 1h each at room temperature (RT) and then incubated overnight at RT on a platform shaker in a solution of PBSG-T [PBS 1X containing 0.2% gelatin (Sigma-Aldrich), 0.5% Triton X-100 (Sigma-Aldrich) and 0.02% Sodium-Azide (Sigma-Aldrich)]. Next, samples were transferred to PBSG-T containing anti-GFP antibodies (AVES, 1:2,000) and placed at 37°C, with rotation at 100 rpm, for 10 days. This was followed by six washes of 1 h in PBSG-T at RT. Samples were then incubated in secondary antibodies (Donkey anti-chicken Alexa-Fluor 647, Jackson ImmunoResearch, 1:500) diluted in PBSG-T for 2 days at 37°C. After six washes of 1 h in PBSG-T at RT, samples were stored at 4°C in PBS until clearing.

To investigate at the confocal microscope the possible internalization of NMP microsponges by 3T3 fibroblasts, NMPs were labeled with fluorescein-5-isothiocyanate (FITC). After culturing the cells for 24 h, they were incubated in plates with FITC-labelled NMP particles (0.25 g/mL) in DMEM 10% serum for 4 h, 24 h, or 48 h. At the end of each time period, the cells were washed with PBS to remove the excess/unbound NMPs and dead cells. The dead and living cells were counted using a Bürker-Türk counting chamber and the percentage of living cells was determined as an estimate of adherent cells after 4 h, 24 h, or 48 h incubation with NMPs. After incubation, cells were fixed and stained with a red fluorescent marker wheat germ agglutinin (WGA) conjugated to Texas Red (10 μg/mL; Sigma-Aldrich) for 15 min at 37 °C to label the cell, which selectively recognizes plasma and Golgi/ER membrane structures. Afterwards, the cells were fixed with cold methanol and stained with Hoescht 33258 (1 μg/mL; Sigma-Aldrich) to visualize the nuclei. Such samples were observed by means of a confocal laser scanning microscopy (Olympus Fluoview 1000, Tokyo, Japan). Confocal X-Y, X-Z, and Y-Z sections in max intensity projection mode (MIP modality) were taken to display three-dimensional images of MPs cell interaction.

Immunohistochemistry experiments were carried out as described previously [11 (link)] on coronal brain slices (50–100 μm, vibratome, Microm; 14 μm, cryostat, Leica) with the appropriate primary (Table A in S1 File) and secondary (Alexa Fluor_568 or 647-conjugated goat anti-rabbit, anti-mouse, anti-rat, anti-guinea pig IgGs or donkey anti-goat IgGs; Life Technologies) antibodies. Hoescht 33258 (1:2000, Sigma) was used for nuclei staining.