Paraformaldehyde (pfa)

Focal demyelination was performed by stereotactic injection of Lysolecithin (LPC) (SIGMA-ALDRICH, St Louis, USA) as described previously (Cayre et al., 2013 (link); Magalon et al., 2007 (link)). The corpus callosum from healthy or demyelinated mice, from the ipsilateral and contralateral side to the LPC-induced lesion, were dissected 7 days post injection (dpi) from 1 mm thick coronal slices in cold Hank's Balanced Salt Solution (GIBCO by life technologie, Paisley, UK) and processed for RT-qPCR analysis (Ndst1 primers : exon 6 (forward, 5′-gctggacaagatcatcaatgg-3′) and exon 7 (reverse, 5′-acacagtacttctacgactatcc-3′); for Gapdh: exon1 (forward, 5’-gggttcctataaatacggactgc-3’) and exon2 (reverse 5’-ctggcactgcacaagaagat-3’). Primers from EUROFINS GENOMICS, Ebersberg, GERMANY). For histological analysis, mice were anesthetized and perfused with ice-cold 4% paraformaldehyde (FISHER SCIENTIFIC, Loughborough Leics, UK) in PBS (GIBCO by life technologie, Paisley, UK). Brains were post-fixed overnight in 4% paraformaldehyde in PBS and cut on a vibratome (Leica) in 4 series of coronal sections (50 µm thick) for immunofluorescence, or cryopreserved and cut with cryostat (20 µm thick) for in situ hybridization.

Mice were randomly separated into two experimental groups (n = 6 per group). Group 1 was composed of 4-month-old animals and group 2 was composed of 25-month-old mice.
The visual cortices of control and aged mice were obtained for IMHQ assays. Briefly, six animals from each group were transcardially perfused with 4% paraformaldehyde (Sigma-Aldrich Chemie, Germany) in 0.1 M phosphate buffer (J.T. Baker, NJ; PB, Tecsiquim; pH 7.4) for the immunohistochemistry assays. Male 4-month-old and 25-month-old C57BL/6J mice were evaluated in the study. Animals were perfused transcardially with phosphate-buffered saline (PBS) and 4% (w/v) paraformaldehyde under sedation. The brains were postfixed in 4% paraformaldehyde for 20 h and immersed in a 30% sucrose solution (w/v) in PBS for 24 h.
Coronal sections (20 μm) from visual cortex were cut on a freezing microtome (Leica CM3050s) and mounted serially. Slides were used for immunofluorescence detection.

After the experiments, the rats were deeply anesthetized with i.p. ketamine and xylazine (120 and 15 mg/kg body mass, respectively). Through the ascending aorta (right atrium cut), the rats were perfused with 100 mL (10 mL.min^-1) of heparinized (10 U.mL^-1) phosphate-buffered saline followed by 400 mL (10 mL.min^-1) of cold 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in PBS using a peristaltic metering pump (Milan, PR, Brazil). The brains were removed, post-fixed in paraformaldehyde for 48 h at 4°C, and transferred to a 30% sucrose solution at 4°C for 48 h. The brains were then cut on a cryostat (Leica Microsystems, Srt. Heidelberg, Germany) into 50-μm sections and stored at 4°C in a 0.9% saline solution until being mounted on glass slides. The brain slices were stained with 0.5% cresyl violet and examined under a light microscope. The positions of the thermistor tips were confirmed by comparing the sites of the brain lesions to the neural substrates described in Paxinos and Watson’s atlas (2007).
As shown in Fig 1 and Table 2, no differences were observed between the groups with regard to the stereotaxic coordinates at which the thermistor tips were positioned. These data indicate that the different T_brain responses observed in the SHRs during exercise resulted from hypertension per se and not from the positioning of the thermistor tips.

Nanofiber scaffolds containing T17b eEPCs were washed with PBS first and fixed in multiple steps, starting with incubation in a 0.2 M sodium cacodylate trihydrate (Sigma Aldrich, Schnelldorf, Germany) buffer, containing 0.1% glutaraldehyde (Carl Roth, Karlsruhe, Germany), 2% paraformaldehyde (Carl Roth), and 5% sucrose (Sigma Aldrich, Schnelldorf, Germany) for 1 h at room temperature. In a second step, samples were placed in a 0.2 M cacodylate buffer, containing 0.3% glutaraldehyde and 3% paraformaldehyde for 1 h, followed by a series of ethanol dilutions in rising concentration, ending at 100 vol.%.
Fixed samples were then critical point dried with EM CPD300 (Leica Microsystems, Wetzlar, Germany), removed from Minusheets, gold-palladium sputtered, and examined using an AURIGA® scanning electron microscope (SEM) (Carl Zeiss, Jena, Germany).

LX-2 cells were purchased from Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China) and seeded on 12-chamber slides (Corning, Kennebunk, USA) at a density of 50,000 cells per well. The next day, TEVs (10 μg/mL) previously stained with DiI (AAT Bioquest, USA) were added to the media for 24 h, then the cells were fixed with 4% paraformaldehyde (Wako, Japan) and nuclei were stained by DAPI (Biosharp, Hefei, China). Finally, the intracellular uptake of TEVs was analyzed using a fluorescence microscope (Leica Microsystems, Wetzlar, Germany).
To study TEVs taken up by liver tissue in vivo, healthy mice were orally administered with DiR-loaded TEVs (1 mg protein/kg) for 24 h, mice were sacrificed and liver tissues were fixed in 4% paraformaldehyde.