Cm1850 cryostat

Fresh TA, soleus (Sol), and EDL muscles were embedded in optimal cutting temperature compound and frozen in isopentane chilled on dry ice. Then, the tissues were cut at 10-μm thickness by Leica CM1850 cryostat (Leica Biosystems, Wetzlar, Germany). Adipose tissues were fixed in 10% formalin for 24 h at room temperature. Then, the adipose tissues were embedded in paraffin and cut into 4-μm thickness slices, deparaffinized, and rehydrated using xylene, ethanol, and water by standard methods. For hematoxylin and eosin (H&E) staining, the sections were stained in hematoxylin (30 min), rinsed in running water, and stained in eosin (1 min). H&E staining images were captured with a Nikon D90 digital camera (Nikon, Tokyo, Japan) mounted on a microscope.

Immediately after the last imaging session, mice were killed, perfused with formalin and brains extracted. The tissue was immediately immersed in formalin. Coronal sections (30 μm) were generated using a Leica CM1850 cryostat (Leica Biosystems, Buffalo Grove, IL). The sections were incubated for 1 h in a blocking solution (0.3% bovine serum albumin (BSA), 8% goat serum and 0.3% Triton X-100) at room temperature, followed by incubation at 4 °C overnight with the following primary antibodies diluted in blocking solution: (1) anti-nestin (Millipore, MAB353, 1:250), (2) anti-GFAP (Millipore, MAB3405, 1:250), (3) anti-Tuj-1 (Sigma, T8578, 1:1,000), (4) CD133 (Millipore, MAB4399, 1:100) and (5) Ki-67 (Dako, M7240, 1:20). The sections were washed three times with PBS, incubated in the appropriate secondary antibody and visualized using a confocal microscope (Olympus). Paraffin embedding, tissue sectioning and haematotoxylin and eosine staining were performed by the UNC Animal Histopathology Core.

After the final ABR test [see Auditory Function Evaluation (Auditory Brainstem Responses, ABR)], animals were deeply anesthetized (ketamine hydrochloride 25 mg/kg, xylazine 5 mg/kg, and acepromazine maleate 1.5 mg/kg body weight) and perfused transcardially with 0.9% NaCl (room temperature), followed by 4% paraformaldehyde at day 7 after surgery. The cochleae were quickly removed under a dissecting microscope (Zeiss Stemi DV4 Stereo Microscope, Jena, Germany) and processed for fluorescence and immunofluorescence analyses (described below). After 24 h of incubation with 4% paraformaldehyde in PBS at 4°C a pH 7.5, the cochleae were decalcified for 15 days (10% EDTA, changed daily), incubated for 48 h in cold 30% sucrose, and embedded in OCT. Frozen tissue samples were then cut into 16 μm thick sections using a Leica CM1850 cryostat (Leica Biosystems, Wetzlar, Germany). Five/ten neighboring randomly selected midmodiolus slices per cochlea were used to analyze the expression of transgenic GFP expression, as a reporter for ASC presence and survival; DAPI labeling was used to identify condensed cell nuclei. The analysis was performed using a fluorescence microscope equipped with a digital camera (Olympus BX63, Tokyo, Japan) and a confocal laser scanning system (TCS-SP2; Leica Microsystem, GmbH, Germany).

To investigate whether lomerizine modulates LPS-induced neuroinflammation in vivo, lomerizine (20 or 30 mg/kg, i.p.) or vehicle (2% DMSO) was administered to wild-type mice (8 weeks old) daily for 7 days. On day 7, LPS (10 mg/kg, i.p.) or PBS was administered 30 min after the lomerizine or vehicle injection. Eight hours later, the mice were sacrificed, perfused with PBS, and fixed in 4% paraformaldehyde (PFA). The brains were postfixed in 4% paraformaldehyde (PFA) overnight at 4°C. After removing the 4% PFA, the brains were stored in 30% sucrose for 72 h at 4°C, frozen at -80°C with OCT compound (Sakura Finetek USA, Torrance, CA), and sliced at a thickness of 30 µm at -23°C using a Leica CM1850 cryostat (Leica Biosystems, Buffalo Grove, IL, USA).

At 24 h after intratracheal administration of SLNas samples, lungs were dissected and flash-frozen with liquid nitrogen in Tissue-Tek O.C.T. Compound. Transverse sections of 6 μm thickness were obtained at various points along the length of the tissue (superior, median, and caudal regions) using a Leica CM-1850 cryostat (Leica Biosystems, Inc., Buffalo Grove, IL, USA). For histopathological evaluation, the sections were stained with H&E and analyzed using light microscopy (DM 2000 Leica Microsystems, Bannockburn, IL, USA) coupled to a photographic camera (Canon PowerShot S80, VA, USA). Moreover, lung sections were observed by fluorescence microscopy (DMI 4000B, Leica Microsystems, Bannockburn, IL, USA).

The animals were sacrificed 5 days (n = 4 per group) or 18 days (n = 4 per group) after FUS sonication. The rats were anesthetized via the intraperitoneal injection of a mixture of ketamine (75 mg/kg) and xylazine (4 mg/kg). For the blood wash-out and brain fixation, transcranial perfusion was performed with 0.9% normal saline and 4% paraformaldehyde in 1× PBS. After perfusion, all brains were post-fixed in 4% paraformaldehyde for 1 h. Subsequently, the brain tissue was transferred to a 30% sucrose solution for 3 days. The brains were then sectioned into 30-μm-thick slices using a Leica CM1850 cryostat (Leica Biosystems, Wetzlar, Germany).