Alexa fluor 488 conjugate anti rabbit secondary antibody

The ivabradine, gelatin, protease inhibitor cocktail, LY294002, dimethyl sulfoxide (DMSO) and 4', 6-diamidino-2-phenylindole (DAPI) were obtained from Sigma (Saint Louis, Missouri, USA). Phosphatase inhibitor cocktail tablets were from Roche Applied Science (Mannheim, Germany). RIPA buffer and Reactive Oxygen Species Assay Kit were purchased from Beyotime (Nantong, China). BCA Protein Assay Kit and SDS-PAGE loading buffer were obtained from Keygen (Nanjing, China). Primary antibodies against GAPDH, phospho-eNOS, phospho-Akt, phospho-rictor, phospho-p70S6K, phospho-S6RP, phospho-raptor, IL-6, VCAM-1 and HRP-conjunct secondary antibody, Alexa Fluor® 488 conjugate anti-rabbit secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Alexa Fluor® 488 conjugate goat anti-mouse secondary antibody was obtained from Thermo Fisher Scientific (Waltham, MA, USA). PVDF membrane and Immobilon western chemiluminescent HRP substrate were obtained from Millipore (Billerica, MA, USA). ROS fluorescent probe-DHE was from Vigorous Biotechnology (Beijing, China). TRIzol was purchased from Invitrogen (Grand Island, NY, USA). PrimeScript RT reagent Kit and SYBR Premix Ex Taq were from TakaRa Biotechnology (Dalian, China).

An original method was applied to label proteins in the glioma neurospheres. Neurospheres were placed into cell insert (Millipore, US) and fixed by 0.4% Paraformaldehyde solution (Solarbio, China). Then, the neurospheres were washed three times in PBS, and incubated with primary antibodies (See Supplementary Table 1) overnight at 4°C. Alexa-Fluor 488 conjugate anti-rabbit secondary antibody (1:5000, Cell signaling, US) and Alexa-Fluor 594 conjugate anti-mouse secondary antibody (1:5000, Life technologies, US) were used for fluorescent double-staining. Serum medium cultured CD133+ cells were stained through Alexa-Fluor 594 conjugate phalloidin (1:200, Life technologies, US) for 20 minutes at 37°C to exhibit actin cytoskeleton. DAPI solution (Solarbio, China) was employed to label cell nucleus. Images were observed and captured by Perkinelmer UltraVIEW VOX confocal microscope (Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China).

Intestinal tissue was paraffin-embedded and cut into five-micron-thick longitudinal sections followed by H&E staining. For immunohistochemistry, the un-stained slides were deparaffinized and blocked with 3% hydrogen peroxide in methanol. Antigen retrieval was performed using Diva Decloaking solution (Biocare Medical, Concord CA) (120 °C for 2 min using a pressure cooker). Slides were blocked with avidin-pink and biotin-blue (Biocare Medical), treated with anti-DCLK1 antibody (#62257, 1:400; Cell Signaling Technology, Danvers MA) in DaVinci Green (Biocare Medical), incubated overnight at 4 °C, and visualized with biotinylated goat anti-rabbit IgG (Jackson ImmunoResearch Inc. West Grove PA) followed by streptavidin-horseradish peroxidase (Jackson ImmunoResearch Inc. West Grove PA), diaminobenzidine (Sigma-Aldrich), and hematoxylin counterstaining. Organoid whole-mount immunofluorescence staining was performed according to published method^{17 (link)} with minor modification. Briefly, the organoid was fixed with 10% neutral-buffered formalin, quenched with 50 mM NH₄Cl, permeabilized with 0.5% Triton X-100, blocked with 5% BSA, treated with DCLK1 antibody (#62257, 1:100; Cell Signaling Technology, Danvers MA) and finally visualized with Alexa Fluor^® 488 Conjugate anti-rabbit secondary antibody (#4412, 1:250; Cell Signaling Technology, Danvers MA).