Sc 390545

AR(rabbit monoclonal, 1:1000, #5153), PSA/KLK3(rabbit monoclonal, 1:1000, #5365), TOMM20(rabbit monoclonal, 1:1000, #42,406), ATG5(rabbit monoclonal, 1:1000, #12994S), LC3I/II(rabbit monoclonal, 1:1000, #12,741), Akt (rabbit monoclonal, 1:1000, #4691), Phospho-Akt (Ser473)(rabbit monoclonal, 1:1000, #4060), Phospho-Akt (Thr308)(rabbit monoclonal, 1:1000, #13,038), Nanog(rabbit monoclonal, 1:1000, #4903), Sox2(rabbit monoclonal, 1:1000, #3579), ALDH1A1(rabbit monoclonal, 1:1000, #36671S) and VDAC(rabbit monoclonal, 1:1000, #4661) antibodies were purchased from Cell Signaling Technology (CST). NCAM1(rabbit polyclonal antibody, 1:1000, A7913), β-Actin(mouse polyclonal, 1:5000, AC004) and α-Tublin(mouse polyclonal, 1:5000, AC012) antibodies were purchased from ABclonal Technology (Upper Heyford, UK). NSE (rabbit monoclonal, 1:1000, ab180943) and Syp (Rabbit monoclonal, 1:1000, ab184176) were purchased from Abcam (Shanghai, China). ALDH1A1(mouse monoclonal antibody, 1:500,sc-166362), TOMM70(mouse monoclonal antibody, 1:500, sc-390545),AR(rabbit polyclonal, 1:1000, Sc-815X) antibodies were purchased from Santa cruz. E-cadherin (rabbit polyclonal, 1:1000, GTX100443),BRN2(rabbit polyclonal, 1:1000, GTX114650) and N-cadherin (rabbit polyclonal, 1:1000, GTX127345) were purchased from Genetex.

The mice were anesthetized with 2% pentobarbital sodium and perfused transcardially with 20 ml of ice-cold 0.1M PBS and 20 ml of ice-cold 4% paraformaldehyde. The brains were removed, fixed in 4% paraformaldehyde, and incubated in 30% sucrose solution overnight at 4°C. Sagittal sections were cut on a freezing microtome at a thickness of 20 μm (Leica Biosystems Inc., Nussloch, Germany). The sections were treated with 1% Triton X-100 (ST795, Beyotime, Shanghai, China), and blocked with 5% normal goat serum (SL038, Solarbio, Beijing, China) for 1 h.
The treated cells were fixed in 4% paraformaldehyde for 20 min, treated with 0.5% Triton X-100 (ST795,Beyotime, Shanghai, China), and blocked with 5% normal goat serum (SL038, Solarbio, Beijing, China) for 1 h.
After blocking with 5% normal goat serum, mouse brain sections and HT22 cells were incubated with primary antibody against TOM70 (1:200 dilution, sc-390545, Santa Cruz, Dallas, Texas, USA) overnight at 4°C and incubated with secondary antibodies (1:500 dilution, ab150115, Abcam, Shanghai, China) for 1 h at room temperature. Finally, the nuclei were stained with DAPI (F6057, Sigma, Shanghai, China), and images were observed using fluorescence microscopy and confocal microscopy.