Hoechst

HK2 cells were seeded in 24-well plates and treated with TNF-α and CHX. Then, the cells were stained with Hoechst (Beyotime, China) and PI (BD, USA). Images were collected with a fluorescence microscope (Carl Zeiss, Germany).

Cell proliferation was determined using an EdU Proliferation Kit (Beyotime, C0071S, Shanghai, China). Cells were cultured in a 48-well plate for 24 h, then incubated with 50 mM EdU solution for 2 h and fixed in 4% paraformaldehyde. Subsequently, the cells were permeabilized with 0.25% Triton X-100 for 15 min and sequentially stained with Alexa Fluor 488 (Lot:12194) and Hoechst (Beyotime, 33342, Shanghai, China).The EdU-treated cells were then imaged and assessed using an Olympus FSX100 microscope (Olympus, Tokyo, Japan).

Cells were planted on 24-well plates chamber slides and grew overnight to adhere. Dual immunostaining was performed sequentially. First, the cells were fixed with cooled 4% paraformaldehyde for 30 min and permeabilized with 0.25% Triton X-100 for 1 h. After being blocked with 5% BSA for 1 h, the cells were incubated in 5% BSA at 4 °C overnight with the primary antibody. Next, the cells were washed with PBS twice and incubated in 5% BSA for 1 h at room temperature with secondary antibodies Alexa Fluor 488 (Lot:12194) and Cy3(Lot:125099) from Jackson ImmunoResearch (USA). Nuclei were stained with Hoechst (Beyotime, 33342, Shanghai, China). Photographs were taken using a confocal microscope (Carl Zeiss Microscopy GmbH, Germany).

Immunofluorescent staining of mature or differentiated NSs was performed as described (Golmohammadi et al. 2008 (link)). The NSs were fixed with 4% paraformaldehyde (Amresco, Solon, OH) for 30 min at RT. After one PBS wash, they were blocked in blocking solution [PBS with 0.1% triton100 (Amresco), 5% FBS, and 5% goat serum (ZSGB-Bio, Beijing, China)] for 60 min at 37°C. After that, the NSs were firstly stained with primary antibody and then with secondary antibody in blocking solution, the nuclei were stained with 10 μg/mL Hoechst (1:1000; Beyotime, Jiangsu, China). Primary antibodies: mouse-anti-nestin (1:200; 307501, R&D, Minneapolis, MN), rabbit-anti-SOX2 (1:200; L1D6A2, Cell Signal Technology (CST), Boston, MA), mouse-anti-GFAP (1:200; GA5, CST) and rabbit-anti-ßIII-tubulin (1:200; D65A4, CST); secondary antibodies: Alexa Fluor 568 donkey-anti-mouse (1:1000; Invitrogen/molecular probe) and Alexa Fluor 488 donkey-anti-rabbit (1:1000; Invitrogen/molecular probe). The coverslips were then mounted using GVA mounting medium (ZSGB-Bio).

To determine the expression of recombinant Ts-NBLsp in vivo, quadriceps femoris of three mice in each group were obtained at 48 h post the first immunization. These tissues were fixed in 4% paraformaldehyde (v/v) in PBS, embedded in paraffin, and cut into 3 µm paraffin sections, and then immunofluorescence test (IFT) was performed as described previously. Briefly, the sections were incubated with 0·1% Triton X-100 in PBS at 4 °C for 1 h, blocked for non-specific protein binding by incubation in 10% goat serum diluted in PBS at room temperature for 1 h, and incubated with mouse anti-Ts-NBLsp mAbs (1:200 dilution) at 37 °C for 1 h. After washing, the sections were incubated with a 1:100 dilution of FITC-conjugated goat anti-mouse IgG (Santa Cruz, USA) at 37 °C for 1 h, and then stained with Hoechst (Beyotime Biotechnology, Beijing, China) for 5 min at room temperature. The sections incubated with serum from an unimmunized mouse at the same dilution served as a negative control. The sections were examined and photographed under fluorescent microscope (Olympus, Japan).