Ts2 fc

First, sterile cell slides were placed at the bottom of 24-well plates, and Eca109 and CaEs-17 cells were seeded on the slides. Then the cell transfection was conducted. After 48 h, the medium was discarded, and cells were fixed with 500 μL pre-cooled fixative for 15 min. After rinsing, 500 μL of 0.5% Triton X-100 was added for cell permeabilization. Then the cells were blocked with 3% BSA. Primary antibodies anti-E-cadherin (ab40772, 1:500) and anti-vimentin (ab92547, 1:500) were added and incubated overnight at 4°C. Next day, fluorescent secondary antibody goat anti-rabbit IgG (ab150077, 1:500) was then incubated with cells at 37°C for 30 min in dark. After rinsing, the cell slides were taken out and dried, then pasted onto the cover glass that containing the mounting medium (with DAPI) (Mesgenbio, China), and the extra fluid was removed after light pressure. Images were taken by inverted fluorescence microscope (Ts2-FC, Nikon, Japan).

The cells with 90% confluence were cultured on 12-well plates. At 48 h after transfection, the cells were processed by the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (C0078s, Beyotime, CHN) according to the instructions, and the slides were observed and photographed under the fluorescence inverted microscope (Ts2-FC, Nikon, JPN).

To prepare brain tissue sections, they were first deparaffinized in xylene and rehydrated through graded ethanol. Antigen retrieval was performed, followed by blocking to prevent non-specific binding. The sections were then incubated with primary antibodies (Tyrosine Hydroxylase (TH), ab137869, Abcam; α-syn, ab212184, Abcam) and secondary antibodies (IgG H&L (HRP), ab97080, Abcam), with PBS rinses in between. DAB substrate was applied for color development, monitored microscopically, and stopped with distilled water. Hematoxylin was used for counterstaining, followed by dehydration, clearing in xylene, and mounting. The stained sections were then ready for microscopic examination and image capture (Ts2-FC, Nikon, Japan).

IF assays were performed on CSCs in 24-well plates, as previously described (Tang et al., 2020a (link)). After 24 h, the cells were sequentially treated with 4% formaldehyde (1004965000, Sigma, Japan), 5% bovine serum albumin (ST023; Beyotime, China), and 0.5% Triton X-100 (X100; Sigma-Aldrich, St. Louis, MO, USA). Then, cells were incubated with anti-CD44 (ab254530, Abcam, UK) and anti-EpCAM antibodies (#46403; Cell Signaling, Danvers, MA, USA) at 4 °C overnight. The next day, cells were incubated with anti-mouse IgG H&L (ab150113; Abcam) antibody at 25 °C for 1 h. Finally, after incubation with DAPI (1:1000, BS097; Biosharp; Heifei city, China), the cells were observed under an inverted fluorescence inverted microscope (Ts2-FC, Nikon, Japan).

Cell mineralization was evaluated using Alizarin Red S staining. After being stimulated with the indicated agents (curculigoside, Ti), MC3T3-E1 cells were fixed in 4% paraformaldehyde for 15 min. Next, cells were stained with 500 μl/well Alizarin Red solution (Solarbio, China) for 20 min at room temperature. Finally, cells were ringed 3 times with ddH2O and imaged under an inverted fluorescence microscope (Ts2-FC, Nikon, Japan).

The brains of rats was immersed in 10% paraformaldehyde for 24 h, and cut into 4 μm coronal slices after embedding with paraffin. The paraffin slices were parched at 60 °C for 1 h and dewaxed with graded ethanol and xylene. Normal goat serum was utilized as a blocking solution for 30 min at room temperature, and the primary anti-VEGF antibody (1:200, cat. no. GB13034, Wuhan Servicebio Technology Co., Ltd. Wuhan, China), as well as anti-CD31 (1:200, cat. no. GB12063, Wuhan Servicebio Technology Co., Ltd. Wuhan, China), were used to incubate the selections at 4 °C for 12 h. Then, the anti-rabbit secondary antibody (1:300, cat. no. GB21301, Wuhan Servicebio Technology Co., Ltd. Wuhan, China) was used to incubate the sections, and the sections were washed by PBS 3 times. Finally, the section was observed using an inverted fluorescence microscope (Ts2-FC, NIKON, Tokyo, Japan) at 400× magnification. The numbers of positive cells in predefined areas were quantified using Image J software. Six different fields per rat were observed, and each group consisted of three rats. All counts were conducted by blinded observers, and three independent experiments were performed.

HK-2 cells (1 × 10⁴) appended to glass coverslips in 24-well plates were rinsed after treatment. Cells were fixed with 4% paraformaldehyde. After rinsing, a blocking reagent (ST025, Beyotime, China) was taken to seal cells for 1 h at 37°C. Then, cells were subjected to anti-Notch1 antibody (1: 150), anti-Jag1 antibody (1: 200), anticollagen I antibody (1: 250), anti-α-SMA antibody (1: 500), and antivimentin antibody (1: 1000) all night at 4°C, followed by the addition of goat antirabbit IgG H&L (Alexa Fluor® 488, 1: 1000, ab150077) or Alexa Fluor® 594 (1: 1000, ab150080) for 120 min at 37°C. Thereafter, DAPI (C1005, Beyotime, China) was employed to stain the nucleus for 5 min. Fluorescence images were acquired utilizing a fluorescence microscope (Ts2-FC, Nikon, Japan) after blocking with the antifade mounting medium (P0128S, Beyotime, China).