Laser scanning microscope

Human RCECs were cultured on chamber slides in 4-well plates to approximately 90% confluence in serum-free medium overnight before experiments. The cells were then treated with PDGF-C (125 ng/mL, Sino Biological Inc., Beijing, China) or VEGF (10 ng/mL, Sino Biological Inc., Beijing, China) for 1 hr. The slides were then washed, fixed in 4% paraformaldehyde for 10 min, and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 5 min. The slides were blocked with 1% bovine serum albumin and 5% goat serum in PBS at room temperature for 1 hr. After incubation with rat anti-JAM-C (CRAM-18 F26) and rabbit anti-ZO1 (ab59720) (Abcam, Shanghai, China) antibodies and secondary antibodies (Alexa Fluor 488 and Alexa Fluor 594, Yeasen, Shanghai, China). The slides were washed and mounted in medium (DAPI Fluoromount G; SouthernBiotech, Birmingham, AL). A negative control staining followed the same protocol except that the anti-JAM-C or anti-ZO1 antibodies. Fluorescence images were captured using a laser scanning microscope (Leica Microsystems, Wetzlar, Germany).

A total of 12 LRRK2 WT and R1441G mice (n = 6 for each group) were used in this study. The mice were anesthetized with 1% pentobarbital and subsequently positioned in a stable stereotaxic device. The right parietal cortex was made at lambdoid suture coordinates: mediolateral (ML) 2.0 mm, anteroposterior (AP) 1.7 mm. For imaging purposes, a slender cranial window measuring 3 mm in diameter was created.
To evaluate the PVS–ISF exchange, 10 μL of 1% FITC-dextran in artificial CSF was intracisternally injected at a rate of 1 μL/min using a microsyringe (BASi, West Lafayette, IN). Immediately before imaging, 0.2 mL of rhodamine B (1% in saline; Sigma-Aldrich) was administered via tail injection to label the vasculature. A laser scanning microscope (Leica Microsystems, Wetzlar, Germany) was used for our study. The laser was operated at 800 nm, and 25× water-immersion objective lens was utilized for imaging. To analyze the clearance of FITC-dextran from brain parenchyma, lateral images up to 100–300 μm below the cortical surface of the xyz stacks (512 × 512 pixels, 2-μm resolution) were collected at a series of time points: 10, 15, 30, 45 and 60 min after the injection.

Bodipy (D3922, Molecular Probes, Carlsbad, Calif, USA) was diluted in DMSO at 1 μg/ml and applied on slides or tissue section for 30 min at RT. All samples were mounted in VECTASHIELD (Vector Laboratories, Burlingame, CA, USA) and covered with glass cover slips No. 1 (VWR).
We examined cell line samples under epifluorescent optics, and digital images were obtained with a laser scanning microscope (Leica Microsystem, Heidelberg, Germany), while frozen section with microscope from Olympus BX53 (Olympus, Tokyo, Japan).

Frozen sections of renal tissue were fixed with 4 % methanol for 20 min at room temperature and permeabilized with Triton-X100 in PBS for 20 min. Sections were blocked with 5 % goat serum for 30 min, incubated with rabbit anti-RNLS antibody (1:100) overnight at 4 °C, and then incubated with fluorescence-labeled goat-anti-rabbit secondary antibody (Beyotime, Shanghai, China) at 37 °C for 1 h in the dark. Finally, the tissue was stained with DAPI for 5 min. A laser scanning microscope (Leica Microsystems, Germany) was used to acquire images to assess the fluorescence signal of RNLS.

The stable cell line was cultured for 12 h before scanned. Cells were first rinsed with phosphate-buffered saline (PBS), then fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100 for 30 min. The cells were then washed twice, each for 15 min. Confocal images were captured using a laser scanning microscope (Leica Microsystems, Germany) using a 63× oil immersion objective (Fig. 1B).

The cells from the stable cell line at passage six were cultured for 12 h before immunodetection. Cells were rinsed with phosphate-buffered saline (PBS) one time and fixed with 4% paraformaldehyde for 30 min. Then, the cells were washed twice for 15 min each before they were permeabilized with PBT (PBS, 1% Triton X-100) and blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature (RT). Confocal imaging was conducted with a laser scanning microscope (Leica Microsystems, Germany) using a 63x oil immersion objective.