Ab150129

Following ultrasound treatment, the mice were euthanized with 20 mL PBS and 20 mL 10% formalin (Sigma Aldrich, St Louis, MO, USA). The brain was extracted from the skull and fixed in formalin overnight, followed by 15% sucrose for 6 hours and 30% sucrose overnight to protect the tissue before frozen sectioning. The brains were embedded in optimal cutting temperature (OCT; Agar Scientific, Stansted, UK) compound and sectioned into sixty 30 µm slices to cover the entire hippocampus using a cryostat (CryoStar NX70; Thermo Fisher, Waltham, MA, USA).
Immunohistochemistry (IHC) of 12 frozen sections from each brain was used to determine which cells (neurons, microglia or astrocytes) were taking up the probes. For neuronal staining: primary recombinant anti-NeuN antibody (1:500 overnight; Ab177487; Abcam, Cambridge, England) and secondary goat anti-rabbit IgG H&L Alexa Fluor® 488 antibody (1:500 for 2 h; Ab150077; Abcam); for microglia staining: primary anti-Iba1 antibody (1:500 overnight; Ab5076; Abcam) and secondary donkey anti-goat IgG H&L Alexa Fluor® 488 antibody (1:500 for 2 h; Ab150129; Abcam); for astrocyte staining: primary GFAP monoclonal antibody (1:50 overnight; 13-0300; ThermoFisher) and secondary mouse anti-rat IgG2a FITC antibody (1:500 for 2 h; 11-4817-82; ThermoFisher).

The pcDNA3.1(−)-TIM-1 and pVAX1-NS1′ were preserved in our laboratory. JEV E gene was inserted into p3×FLAG-CMV-7.1. hTIM-D114A-His, hTIM-N115A-His, and hTIM-MD were cloned into pcDNA3.1 (+). hTIM-IgVD was cloned into pCAGGS. hTIM-ECD was inserted into prokaryotic expression vectors pET-28a (+) and pGEX. Mouse anti E monoclonal antibody was kept by our lab. Mouse anti-human TIM-1 monoclonal antibody (MAB1750, R&D systems, Minneapolis, MN, USA) and goat anti-TIM-1 polyclonal antibody (AF1750, R&D systems, Minneapolis, MN, USA) were used for detecting the TIM-1 protein in Western blot and IFA. Mouse anti-His antibody (66005-1-Ig, Proteintech, Rosemont, IL, USA) was used for the enrichment of TIM-1-His protein. Mouse anti-GAPDH polyclonal antibody (sc-25778, Santa Cruz, Dallas, TX, USA) was used for Western blot. Rabbit anti-JEV E polyclonal antibody (GTX125867, GeneTex, Irvine, CA, USA), rabbit anti-JEV NS3 polyclonal antibody (GTX125868, GeneTex, Irvine, CA, USA), and rabbit anti-JEV polyclonal prM antibody (GTX131833, GeneTex, Irvine, CA, USA) were used to detected the JEV E, NS3 and prM protein, respectively, in Western blot analyses. Donkey anti-goat IgG-Alexa Fluor488 (ab150129, Abcam, Cambridge, UK), donkey anti-rabbit IgG Alexa Flour 594 (ab150076, Abcam, Cambridge, UK), and donkey anti-mouse Alexa Flour647 (ab150107, Abcam, Cambridge, UK) were used for IFA.

We also analyzed the phenotype of microglia at day 4–5 after isolation. Previously, light microscopy images were taken before staining with antibodies using an AxioObserver Z1 (Carl Zeiss, Jena, Germany). For immunocytochemistry (ICC), the cells were fixed in 4% buffered formalin, washed with 0.1% Triton X-100 in PBS and stained with GFAP (ab16997, Abcam, Cambridge, UK), Iba1 (ab5076, Abcam), TNF-α (ab199013, Abcam) and TGF-β (ab92486, Abcam) antibodies at a working dilution of 1:100 for 1.5 h at RT, then washed and stained secondary antibodies. For staining the appropriate Iba1, Alexa Fluor 488 conjugated anti-goat secondary antibodies ab150129 (Abcam), and for staining the appropriate GFAP, TNF-α and TGF-β, Alexa Fluor 647 conjugated anti-rabbit secondary antibodies A-31573 (Invitrogen, Waltham, MA, USA) were used. After incubation for 30 min at RT, the cells were counterstained with 40,6-Diamidino-2-phenylindole (DAPI) (10 mg/mL in PBS, Sigma, St. Louis, MI, USA) to visualize the nuclei. The results were analyzed using an LSM 780 Confocal Microscope (Carl Zeiss, Jena, Germany).

3 randomly selected, formalin-fixed tissues from Saline- and Au@Ag-treated groups were routinely embedded in paraffin, then 4 mm thick paraffin slices were cut. Samples were deparaffinised, then heat-mediated antigen retrieval was performed using citrate buffer at pH = 6. Samples were blocked with donkey serum (Millipore) and stained with alphaSMA-specific antibody (1:200) (abcam, ab21027) then Alexa488-labelled donkey-anti-goat secondary antibody (abcam, ab150129) was applied. After alphaSMA staining, samples were blocked again using normal goat serum, and Ki67-specific antibody (abcam, ab15580) was used in 1:50 dilution followed by Dylight549-labelled goat anti-rabbit secondary (abcam, ab96984). After immunoreactions, samples were counterstained with DAPI and analysed by an Olympus BX51 fluorescent microscope.
For correlation analysis, 4–5 photographs were taken from the subcutaneous regions of each tumour, then the percentage of alphaSMA- and Ki67-positive cells was quantified using CellProfiler 2.2.0 software. Statistical evaluation of the obtained data was performed using GraphPad Prism 7 software by calculating the Pearson r value.

Differentiated cells were fixed for 30 min with 4% paraformaldehyde, and blocked with 5% normal goat serum and 1% BSA in 0.2% Triton X-100 for 45 min. Primary antibodies were diluted in 5% normal goat serum and incubated with the samples overnight at 4°C. The appropriate fluorescently labeled secondary antibodies were applied for 2 h at room temperature. The nuclei were counter stained with 4, 6-diamidinodiamidino-2-phenylindole (DAPI, 10 mg/ml, Life Technologies). Negative control (omit primary antibody) was included in all immunofluorescent staining. Immuno labeled cells were viewed and counted using Zeiss LSM 710 NLO laser scanning confocal microscope (Jena, Germany). The percentage of MAP-2/TH/DAPI positive cells was calculated within 10 randomly selected visual fields. The following primary antibodies were used: 1:500 rabbit anti-TH (Millipore, AB5935), 1:500 mouse anti-MAP2 (Abcam, ab11267) 1:200 goat anti-GIRK2 (Abcam, ab65096). The secondary antibodies were as follows: Alexa Fluor 488 goat anti-mouse (1:400, ab150113, Abcam), Alexa Fluor 488 donkey anti-goat (1:400, ab150129, Abcam) and Alexa fluor 594 goat anti-rabbit (1:400, ab150080, Abcam).