Ti2 e

The produced GVs were dropped (8 μL) on a microscope slide with a silicon imaging spacer (1 well, diam. × thickness 9 mm × 0.12 mm, Sigma–Aldrich, St. Louis, MO, USA), which were ultra-thin adhesive spacers peeled and stuck to slides to confine specimens without compression. The sample was then covered with a coverslip and left to stand for 10 min to allow the sediment of vesicles to travel to the bottom of the chamber by gravity. GV samples were observed with a Ti2-E inverted microscope (Nikon Ti2-E, Yokohama, Japan), and images were acquired using a confocal microscope (Nikon C2plus, Yokohama, Japan). The green fluorescence of calcein inside the GVs was excited by using a 488 nm laser with emission collected at 498–535 nm. Image analysis was performed using NIS-ELEMENTS C-ER software (Nikon, Yokohama, Japan).

Glioma cells were respectively positioned on glass coverslips (0.17 mm thickness, 14 mm diameter) in a 6-well plate at room temperature overnight. Then cells were washed by PBS, fixed by 4% paraformaldehyde for 30min, infiltrated by 0.1% Triton X-100 for 5min, and blocked by 2% bovine serum albumin (BSA) for 30min in sequence. Incubated with Specific primary antibodies: anti-SNAP25 (1:400; Rat# ab5666;Abcam), anti-GLS (1:400; Rat# ab156876;Abam), anti-MAP2 (1:400;Rat# ab5392; Abcam) at 4°C overnight and rinsed by PBS 3 times, fluorescent secondary antibodies (Donkey anti-Rabbit IgG (H+L) Highly Cross-Absorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, catalog# A-21206, RRID AB_2535792) were applied to specimens and incubated at 37°C in the darkness for 1h. Mounting medium with DAPI DNA counterstain was applied to the specimens followed by images capture (Nikon, Ti2-E).

Human cartilage tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and made in sections. Mouse knee joints were fixed in 4% paraformaldehyde, decalcified in 0.5 µM EDTA, and embedded in paraffin. For Safranin-O and fast green staining, all sections were deparaffinized in xylene, hydrated with graded ethanol, and stained with Safranin-O and fast green (Sigma). Cartilage destruction, synovitis, and osteophyte formation were scored by three observe under blinded conditions using the OARSI (grade 0–6), synovitis (grade 0–3), and osteophyte (grade 0–3) scoring system. The OARSI, synovitis, and osteophyte scores are presented as the mean of the maximum score in each mouse, and each representative Safranin-O-stained image was selected from the most advanced lesion among serial sections. For immunohistochemistry, the slides were treated with EDTA repair solution in a boiling water bath for 30 min, and blocked in 5% BSA (MCE) for 1 h. Primary antibodies against MMP3 (1:100; Abcam, ab52915), MMP13 (1:100; Proteintech, 18165-1-AP), NFKB1 (Proteintech, 14220-1-AP), and p-p65 (Cell Signaling, 3033) were added into the slides at 4 °C overnight. The next day, the slides were washed with PBS thrice and incubated with secondary fluorescence antibodies for 1 h at room temperature. DAPI was used to stain the nuclei. Nikon Ti2-E was used to acquire the fluorescence images.

LLC-PK1 cells were grown in glass coverslips for 24 h, then, co-transfected with plasmids encoding the chaperone RAP and mMeg using Lipofectamin 2000. For the colocalization experiments wild-type mMeg, mMeg S/D or mMeg S/A were used. To visualize Rab11 the receptor was co-transfected with mCherry -Rab11. After 24 h of expression, cells were treated with 50 μM YU142670 or vehicle for 4 h. Cells were fixed with 4% paraformaldehyde in PBS and then permeabilized with 0.2% Triton X-100 in PBS. Next, the cells were blocked with 5% BSA in PBS and incubated successively with the primary antibodies (anti-HA and anti-EEA1 or anti-Rab7) and the corresponding secondary antibodies. Images were captured by Inverted Nikon Ti2-E and deconvolved with DeconvolutionLab (Sage et al., 2017 (link)) Manders coefficient was calculated with JaCoP (Bolte and Cordelières, 2006 (link)), a plugin for ImageJ (NIH). Briefly, images of cells with the two stains were selected and separated. Cells were analyzed with the JaCoP function of Manders’ Coefficient and data was stored for analysis.

Normally, P2 human primary chondrocytes were selected for this experiment. Briefly, the sterile cell plate is carefully put into the 24-well plate, and human chondrocytes were seeded on sterile glass coverslips at the cell density of 3 × 10⁴ per well. When cells were treated with relevant stimulation, growth medium was aspirated and 4% paraformaldehyde was added to fix the cells for 20 min at room temperature. After washes with PBS, 0.2% Triton X-100 was added for 5 min at room temperature to permeabilize the cells, followed by 5% BSA for blocking for 30 min. Primary antibody (1:200) against ENO1 (Proteintech, 11204-1-AP), Aggrecan (Proteintech, 13880-1-AP) and Collagen II (Abcam, ab34712) was added to incubate with the cells overnight at 4 °C. After cells were washed, fluorescent Alexa Fluor^® 555-conjugated anti-rabbit IgG (1:500; Cell Signaling, 4413) was used to incubate for 1 h in dark at room temperature. The cell nuclei was stained by 4′, 6-diamidino-2-phenylindole (DAPI). Images were obtained using Nikon Ti2-E at wavelengths of 555 nm (red) and 405 nm (blue, DAPI).

Immuno-histochemical assays were used to determine CD163 and iNOS protein expression in the bladder tissue of rats. Tissues were obtained from rats with/without HPP treatment, fixed in 10% formaldehyde for 24 h, and then embedded in paraffin. The specimens were cut into 5-μm sections and roasted at 60°C for 2 h. Antigenic retrieval was performed in citric acid buffer (pH = 6.0) followed by washing with PBS and treatment with 3% H₂O₂ and 5% bovine serum albumin. Afterward, the sections were incubated with primary antibodies against CD163 and iNOS (dilutions of 1:50; Cell Signaling Technology) at 4°C overnight. Subsequently, they were incubated with secondary antibody for 30 min. Detection was performed using 3,3′-diaminobenzidine (DAB) chromogen (Maixin Biotech. Co. Ltd, Fuzhou, China), and the sections were counterstained with hematoxylin, dehydrated through graded alcohols, and cleared in xylene. Finally, pictures were taken under 200× magnification under a microscope (Ti2-E; Nikon). Immunostaining was evaluated by Image-Pro Plus 6.0 image analysis software (Media Cybernetics, Inc., Silver Spring, MD, USA).