Hoechst 33342 p0133

Orthotopic tumors were dehydration and sliced to 8 μm by Leica 1950 under −22 ^oC after after treated with M1 at a dose of 16 mg/kg for 48 h. The histologic section was incubated with Anti-CD31 antibody (ab76533, abcam) at 4 ^oC overnight, then washed by TBST and incubated with Goat anti-Rabbit IgG F(ab’)2 Secondary antibody, FITC (31573, Thermo Fisher) for 1 h and washed by TBST. The histologic section then treated by antifade mounting medium with Hoechst 33342 (P0133, Beyotime) and imaged by Perkin Elmer Operetta HCS under 20 x and 40 x with 0 or 2% overlaying.

For immunofluorescence studies, 5×10⁴ cells/well were seeded on the confocal dish (BS-20-GJM; Biosharp, Hefei, China). After 48 h, cells were treated and fixed with 3.7% paraformaldehyde, washed with PBS, and permeabilized with Triton X-100 for a half-hour, followed by incubation blocking solution (1% BSA) for 1 h. Cells were incubated with the following antibodies diluted in PBS with 1% BSA overnight at 4°C: anti-TFEB (dilution 1:500) and anti-ABCA2 (dilution 1:100). The cells were washed with PBS and incubated with rhodamine (TRITC) goat anti-rabbit IgG (H+L) (AS040, ABclonal, Wuhan, China) for 1 h at room temperature. Cell nuclei were stained with Hoechst 33342 (P0133, Beyotime Biotechnology, Shanghai, China). Labeled cells were examined under the ZESIS LSM880 microscope with a ×100/1.4 objective lens. Confocal microscopy images were acquired and processed with LSM880 system confocal microscope software.