Confocal laser scanning microscopy

The immunofluorescent assay were carried out according to procedures performed by us [24 (link), 25 (link)]. Briefly, brains were placed in fresh 30% sucrose solution for 24 h, then embedded in OCT compound (Sakura Finetek, Torrance, CA, USA) and cut at a thickness of 40 μm on a cryostat (CM1950; Leica, Mannheim, Germany). Brain sections were then incubated with following antibody for 1 h in room temperature: glial fibrillary acidic protein (GFAP; 1:400), a marker of astrocytes and ionized calcium binding adaptor molecule 1 (Iba-1; 1:400), a marker of microglia/macrophage-specific calcium-binding protein. After washing 30 min with PBS, then sections were incubated with Dy Light 488-conjugated goat anti-rabbit and goat anti-mouse (1:400; Jackson Immuno Research Labs) antibody for 1 h in room temperature, and washed three times with PBS. Stained sections were captured under a confocal laser scanning microscopy (Nikon Corporation, Tokyo, Japan). Optical density (OD) of immunoreactive structures were measured using ImageJ software (developed at the National Institutes of Health), similar method as previously have described by us [24 (link)].

Prediction of protein localization using the web-based tool (https://psort.hgc.jp/, accessed on 20 October 2019). MfWRKY70 was cloned into a pHB-YFP vector fused with Yellow fluorescent protein (YFP). Sequence-verified recombinant plasmids 35S::MfWRKY70-YFP and 35S::YFP control vector were respectively transferred into Agrobacterium tumefaciens, then injected into the four weeks old tobacco (benthamiana) cells. After 16 h dark treatment and two days of cultivation, the YFP expression in tobacco cells was observed with a confocal laser scanning microscopy (Nikon, Tokyo, Japan).

Ultrapure water was prepared using a Milli-Q water purification system (electrical resistivity of 18 MΩ cm⁻¹). The fluorescence intensity was measured at 25 °C using an LS55 luminescence spectrometer (PerkinElmer, Wokingham, UK) in a quartz cell. The excitation and emission slits were both set with a bandpass of 10.0 nm. A U-1500 UV-vis spectrophotometer (Macy, China) was used to determine the absorbance of biofilms stained by crystal violet (CV). Confocal laser scanning microscopy (CLSM) (Nikon, Japan) and atomic force microscopy (AFM) (Bruker, USA) were used to analyze the distribution and thickness of the biofilm formed on the coverslips.

Confocal laser scanning microscopy (Nikon) was used to observe the internalization of Ara-C@HFn. Before observation, Cy5-labeled Ara-C@HFn was prepared. Briefly, Cy5NHS ESTER (Meilunstar) was dissolved in water and incubated with Ara-C@HFn at a molar ratio of more than 20:1. The mixed solution was stirred at 4 °C overnight, and the free dye was removed by ultrafiltration. Then, HL-60 or K562 cells were incubated with Cy5-labeled Ara-C@HFn for 24 h at 37 ℃ (in RPMI-1640 media) and fixed in 4% cold formaldehyde for approximately 15 min. Subsequently, the cell membrane was stained with rhodamine phalloidin (Invitrogen) for 30 min, and the nucleus was stained with Hoechst 33342 (Solarbio) for 20 min. Finally, the prepared samples were observed with a confocal laser scanning microscope (Nikon) by NIS Elements AR 5.20 (n = 3). Note that for lysosomal staining, it is not necessary to fix the cells before staining. After incubating the lyso-Tracker Green (Beyotime) and Hoechst 33342 (Solarbio) with cells for 20 min at 37 ℃, then the prepared samples could be observed with a confocal laser scanning microscope.

BMMs were seeded into 96‐well plates and treated with different concentrations of tetrandrine in the presence of 20 ng/ml M‐CSF for 3 days and then treated with 20 ng/ml M‐CSF and 50 ng/ml RANKL for 5 days, the cells were fixed by paraformaldehyde (4%) for 15 min at room temperature. After being washed with PBS three times, cells were permeabilized with 0.3% Triton X‐100 for 5 min and blocked with 3% BSA in PBS. Stain the F‐actin rings with rhodamine‐conjugated phalloidin (Eugene, OR, USA) and the cell nuclei with DAPI. Then, capture the images by confocal laser scanning microscopy (Nikon, Tokyo, Japan). The number of multinucleated cells (>3 nuclei) and the number of nuclei were calculated.

Cell proliferation ability was assessed using 5-bromodeoxyuridine (BrdU). Cells were seeded in 3 replicates in 48-well plates with a 6 mm-diameter cover glass in each well. Cells were cultured to approximately 70% confluency and treated with or without 2.5 μg/ml dasatinib for 24 h. Cells were incubated with 10 μM BrdU (Sigma, USA) for 60 min and fixed in 4% paraformaldehyde for 15 min. Fixed cells were permeabilized with 0.1% Triton X-100 and incubated with 2 N HCl for 30 min at 37°C and 0.1 M borate buffer for 10 min at room temperature. Cells were blocked with goat serum and incubated with an anti-BrdU antibody (BD Biosciences, USA, 1:100) at 4°C overnight. Cells were reacted with TRITC-labeled anti-rat IgG. The fluorescent signal was captured using confocal laser-scanning microscopy (Nikon, Japan).