Immunostaining blocking solution

Brains were sectioned coronally at 30 µm thickness with a freezing microtome. One out of every four brain sections was collected in a 0.1 M phosphate-buffered saline (PBS) [34 (link)]. Standard immunofluorescence staining for c-Fos was performed on brain sections from selected brainstem sites. The sections were rinsed three times in PBS on a shaker for 8 min per rinse. The sections were incubated in PBS containing 1% Triton-X 100 for 30 min and rinsed again in PBS containing 0.3% Triton-X 100 for 5 min. After rinsing, the sections were blocked with an immunostaining blocking solution (Beyotime) for 1 h. Sections were then incubated in a rabbit anti-c-Fos primary antibody solution (Abcam, dilution 1:800) for 48 h at 4 °C. Then, the sections were rinsed in PBS on a shaker for 10 min per rinse and incubated with an Alexa Fluor 488-conjugated donkey-anti-rabbit secondary antibody solution (Abcam, dilution 1:800) for 1.5 h at room temperature. Finally, the sections were rinsed three times in PBS on a shaker for 10 min per rinse and counter stained with DAPI (SouthernBiotech). If a represented section of an area was lost during sectioning, the sample was excluded from the study. Therefore, our sample numbers varied slightly between different brain areas, and the sample number per brain region was less than 5 (n ≤ 5).

Circ-GALNT16-specific Cy3-labeled probe was used to detect the subcellular localization of circ-GALNT16 in DLD-1 and LoVo cells by using a FISH Kit (RiboBio). Briefly, after the cells were fixed with paraformaldehyde for 10 min and permeabilized for 5 min by using PBS with 0.5 % Triton X-100, a FISH probe hybridized with a preheated hybridization buffer was mixed with cells and incubated overnight at 37 °C. The cells were then washed with 4× sodium citrate buffer containing 0.1 % Tween-20 for 5 min and 1× SSC for 5 min. The cell nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI). The images were obtained using a confocal fluorescence microscope.
For the dual RNA-FISH and immunofluorescence assay, an immunostaining blocking solution (Beyotime) was used for cell blocking for 1 h after incubation with the FISH probe as described above. The cells were then incubated with hnRNPK antibodies overnight and labeled with fluorescent secondary antibodies for 1 h in the dark. Finally, DAPI was used for nuclear staining.

To evaluate the macrophage-platelet interaction during DBV infection, 8 × 10⁵ PMA-activated THP-1 cells were seeded onto a confocal plate (Biosharp, Anhui, China) and infected by DBV (MOI = 1). Macrophages treated with LPS and nigericin or untreated were served as positive and negative controls, respectively. After 72 h incubation, macrophages were washed three times with precooled PBS, fixed with 1% paraformaldehyde for 5 min, and penetrated with 0.1% Triton-X for 10 min. After blocking with immunostaining blocking solution (Beyotime, Shanghai, China) for 10 min, samples were incubated overnight at 4°C with anti-tubulin antibody (1:200) (Abcam, Britain), anti-caspase-1 antibody (1:200) (Santa Cruz Biotechnology, USA) and anti-DBV-Gc antibody (1: 200) (Sangon Biotech, Shanghai, China). Samples were washed with PBS and incubated with donkey anti-rabbit IgG H&L (Alexa Fluor 568) pre-adsorbed (1:200) (Abcam, Britain), goat anti-mouse IgG H&L (Alexa Fluor 488) pre-adsorbed (1:200) (Abcam, Britain) and donkey anti-rat IgG H&L (Alexa Fluor 647) pre-adsorbed (1:200) (Abcam, Britain) for 1 h. The nuclei were then stained using the mounting medium with DAPI (Abcam, Britain). The images were obtained using a laser confocal microscope (ZEISS, LSM880, Germany).

RNA FISH was conducted to assess the subcellular localization of circ_0053943 in MUM2B, C918, and OCM-1A cells using a FISH Kit (RiboBio) following the manufacturer’s instructions. Circ_0053943-specific Cy3-labeled probes were designed and synthesized by RiboBio. Briefly, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in phosphor-buffered saline (PBS), and blocked with a prehybridization buffer. Subsequently, the cells were incubated in a hybridization buffer containing a FISH probe overnight. After rinsing with sodium citrate buffer, cells were incubated with Hoechst 33342 (ThermoFisher Scientific, MA, USA) for nuclear staining. For dual RNA-FISH and immunofluorescence, cells were blocked with an immunostaining blocking solution (Beyotime) after incubation with the hybridization buffer containing the FISH probe. Subsequently, cells were incubated with primary antibodies overnight and labeled with fluorescence-conjugated secondary antibodies for 1 h in the dark. Hoechst 33342 (ThermoFisher Scientific) was added for nuclear visualization. Images were acquired using the Nikon A1Si Laser Scanning Confocal Microscope (Nikon Instruments, Tokyo, Japan).

A population of 5.0 × 10⁴ cells was seeded onto the electrospun fibers and cultured in the incubator for 24 h. The cells were then subjected to three washes with PBS and subsequently fixed in a 4% paraformaldehyde solution for 30 min. After another round of PBS washing, the cells were incubated in an immunostaining blocking solution (Beyotime Biotechnology, China) for 1 h to facilitate closure. Next, rabbit‐derived NGF monoclonal primary antibody (Abcam, Cambridge, UK) and rabbit‐derived SOCS3 monoclonal primary antibody (Abcam, Cambridge, UK) were added and allowed to incubate overnight at 4 °C in a refrigerator. Following a thorough wash, RBITC and CY3‐labeled secondary antibodies (Beyotime Biotechnology, China) were separately introduced. After another round of washing, FITC‐labeled peptide (Beyotime Biotechnology, China) was added and allowed to incubate for 40 min to label the cell membrane. Finally, DAPI (Beyotime Biotechnology, China) was added and incubated for 5 min to enable observation of protein expression under a fluorescence microscope (Leica, Germany). Images were captured for subsequent calculation of protein expression levels.