Cm1950

For uterine tissue retrieval, mice were anesthetized with 1 % sodium pentobarbital (50 mg/kg, i.p.), and perfused transcardially with 10–15 mL phosphate-buffered saline (PBS) followed by 10–15 mL of ice-cold 4 % paraformaldehyde (pH 7.4). We performed a laparotomy via a lower abdomen midline incision to dissect and remove the uterus. Then we post-fixed the uterus in 4 % paraformaldehyde in PBS for 3 h and stored it overnight in 30 % sucrose for cryoprotection. The uterus was then embedded in optimum cutting temperature compound (OCT, Leica, Wetalar, Germany) and rapidly frozen at −20 °C (CM1950, Leica). Cryoembedded tissues were cut into 10-μm thick slices on a sliding microtome (CM1950, Leica). Conventional H&E-staining technique was used to confirm the distribution of Pirt nerve fiber terminals on the uterus. All imaging was performed with an Olympus fluorescence microscope (BX51, Olympus, Shinjuku, Tokyo, Japan).

Immunofluorescent imaging of TG and skin tissue were performed as previously described 20 (link). Briefly, mice were anesthetized with 1% sodium pentobarbital (50 mg/kg, i.p.) and transcardially perfused with cold 0.1 M phosphate-buffered saline (PBS, pH 7.4, 4℃) followed by 4% paraformaldehyde in PBS (pH 7.4, 4°C). TG and skin were then dissected and cryoprotected in 30% sucrose at 4°C for 24 hours. Afterwards, dissected tissues were embedded in optimum cutting temperature compound (OCT, Leica, Wetalar, Germany) and rapidly frozen at -20°C (CM1950, Leica). TGs and skin were sectioned at 10 μm or 50 μm thickness, respectively, and slide mounted using a sliding microtome (CM1950, Leica). Slides were then incubated in blocking solution (10% fetal bovine serum in PBS containing 0.1% Triton X-100) for 30 min at room temperature, followed by primary antibody at 4°C overnight and in secondary antibody at room temperature for 2 hours in the dark. Images were acquired using an Olympus fluorescence microscope (BX51, Olympus Japan).

Muscle fiber cross‐sectional area (CSA) analysis was performed in the tibialis anterior (TA) muscles of sham and stroke mice. The TA muscles were collected, mounted to cork blocks using tissue freezing medium (OCT, Thermo Fisher Scientific), flash frozen using isopentane cooled in liquid nitrogen, and then stored at −80°C. Frozen tissue cross sections measuring 10 μm thick were cut from the mid‐belly of the muscles using a cryostat (Leica CM1950, Leica CM1950), mounted on charged microscope slides (Fisher Scientific), and stored at −80°C. For staining, the slides were air‐dried 10 minutes, hydrated in 1× PBS for 5 minutes, and incubated for 60 minutes in 10% normal goat serum (Vector laboratories) at RT. The tissue sections were then incubated overnight at 4°C with the rabbit anti‐Laminin primary antibody (Sigma). After washing three times in 1× PBS, the sections were incubated in anti‐rabbit Alexa Fluor 555 secondary antibody (Thermo Fisher Scientific) for 1 hour at RT. After washing in 1× PBS for three times (5 minutes each), the sections were mounted with Prolong Gold antifade mounting media (Thermo Fisher Scientific) for imaging with a Zeiss LSM 510 Meta confocal microscope confocal microscope at WVU imaging facility. Muscle fiber CSAs were traced and measured manually (∼300 fibers per muscle) in ImageJ v.1.43.

In each of the 4 groups, 3 mice were used for haematoxylin-eosin staining (H&E) at 3 days post-CCI. The animals were euthanized by intraperitoneally injecting sodium pentobarbital (65 mg/kg), then perfused transcardially with phosphate-buffered saline followed by 50 mL of 4% paraformaldehyde. The brains were quickly dissected from the mouse body and fixed in 4% paraformaldehyde at 4 °C for 48 h. Coronal sections, which were obtained using a vibratome (Leica VT 1000S, Wetzlar, Germany), should contain the entire hippocampus (–0 mm, –3.5 mm relative to bregma). Serial coronal sections (30 μm thick) for H&E staining (n = 3 per group) were cut by a cryostat (Leica CM 1950). At the same time, the colon tissue was excised and fixed in a 4% paraformaldehyde solution, dehydrated using ethanol and xylene, and embedded in paraffin. Moreover, 5 mm thick sections were cut for H&E staining. In addition, the injured cerebral cortex (respective n = 3) and colon tissue (respective n = 3) were quickly removed, weighed, frozen in liquid nitrogen, and then stored at −80 °C for Western blotting, and the cerebral cortex on the injured side (respective n = 3) was used for RNA-Seq. Furthermore, the faecal samples (respective n = 3) were quickly collected, weighed, frozen in liquid nitrogen, and then stored at −80 °C for SMRT sequencing.

Model fibrous microplastics were prepared using the method initially developed by M. Cole [43 (link)]. Briefly, three types of textile-relevant fibers, including PES and PAN (Reinhard Strauss GmbH & Co. KG, Viersen, Germany) and PA6 (Heimbach Group GmbH, Düren, Germany), were aligned, embedded in a water-soluble freezing agent (Tissue-Tek^® O.C.T. Compound), and sectioned into microfibers with a length of 10 μm. The sectioning was performed using a cryotome (Leica CM1950). The freezing agent was washed off and powders of microfibers were obtained using lyophilization. The resulting particles were imaged via a Hitachi S3000 scanning electron microscope (SEM) and their size distribution was analyzed using ImageJ.

After behavioral testing or CSF tracer injection, anesthetized mice were transcardially perfused with 50 mL 0.9% saline, followed by 60 mL 4% PFA for 5 minutes by a perfusion pump (Longer Pump, Cat # BT100‐2J). Brains were then removed and postfixed in 4% PFA at 4°C overnight. For immunohistochemical staining, postfixed brain tissues were dehydrated in a series of graded ethanol solutions and embedded in paraffin. Coronal sections containing the hippocampus were cut at 5 μm using a paraffin slicing machine (Leica RM2135). For double immunofluorescence, forebrains were dehydrated gradiently in 20% and 30% sucrose solutions for each 24 hours at 4°C. Tissue was then embedded in Tissue‐Tek OCT compound to be made into coronal sections of 30 μm thickness, using a freezing microtome (Leica CM1950). For CSF tracer experiments, PFA postfixed forebrain tissues were sliced with a vibrating microtome (World Precision Instruments Inc, WPI; Cat. # NVSLM1‐436), at a thickness of 100 μm, and mounted onto gelatin‐coated slides in sequence.

The histology and immunostaining were conducted as previously described (Xie et al., 2018 ; Tang et al., 2016a (link)). After pentobarbital overdose, mice were intracardially perfused with 0.9% saline solution followed by 4% paraformaldehyde in PBS. Mouse brains were removed immediately and placed in 4% paraformaldehyde solution overnight for post-fixation and then soaked in 30% sucrose solution for at least 48 h for cryoprotection. With a cryostat (Leica CM1950), the brains were sequentially cut into 40-μm-thick coronal sections from the olfactory bulb to the cerebellum (approximately +4.50 mm to −6.40 mm from the bregma). The sections were cleaned with PBS and mounted on chrome-gelatin subbed glass slides in sequence. To enhance the fluorescence intensity of the HSV-EGFP labeled slice, the sections were blocked with 3% BSA in PBS-0.3% Triton X-100 for 1 h at 37°C and subsequently incubated with anti-GFP antibody conjugation FITC (1:400, Abcam, ab6662) for 2 h at 37°C. After washing, brain sections were coverslipped with 70% DAPI-glycerol mounting medium.