Fluorescent mounting solution

Freshly obtained tumor tissues were fixed using 2% paraformaldehyde, and then infiltrated with sucrose and embedded using the OCT compound. For immunostaining, the sections were blocked with bovine serum albumin and stained with anti-CD31, anti-CD44, and anti-CD24. The samples were mounted using fluorescent mounting solution (DAKO).
To assess the differentiation capacity of the organoids, differentiated organoids and control organoids were stained as previously described, with minor modification 13 (link). In brief, the differentiated and control organoids were fixed in paraformaldehyde and glutaraldehyde for 10 min. Then the organoids were washed with phosphate buffered saline containing 10 mM NaBH4 and stained with anti-carbonic anhydrase II (CA2) (Santa Cruz Biotechnology). The organoids were stained with a secondary antibody and 4,6-diamidino-2-phenylindole. Fluorescence images of the stained organoids and frozen sections were obtained using a Zeiss LSM710 laser scanning confocal microscope.

The primary antibodies used for immunocytochemistry were: mouse monoclonal anti-nestin (MAB5326; Millipore), mouse monoclonal anti-GFAP (ab4648; Abcam), mouse monoclonal anti-Tuj1 (MAB1195; R&D Systems, Minneapolis, MN, USA) and mouse monoclonal anti-P53 (2524; Cell Signaling Technology Inc.). Both fluorescent secondary antibody conjugates, goat anti-mouse Alexa Fluor 488 (Invitrogen) and goat anti-rabbit Alexa Fluor 488 (Invitrogen), were used at a 1:1000 dilution. DAPI was used for counterstaining. The coverslips were mounted with fluorescent mounting solution (DAKO, Carpinteria, CA, USA) and observed by confocal microscopy (Eclipse TE200, Nikon, Japan).

Autophagy was assessed by LC3 lipidation and GFP-LC3 redistribution. For GFP-LC3 dot formation, stable GFP-LC3-expressing AML cells were cytospun, fixed with 4% paraformaldehyde for 20 min at RT, washed with PBS, and covered with fluorescent mounting solution (Dako) prior to analysis by confocal microscopy (LSM510, Carl Zeiss). To quantify GFP-LC3 dots, at least 100 cells per slide in three independent experiments were assessed, that is, the percentage of GFP-LC3-positive cells with punctuate staining; and the number of discrete puncta per cell was counted.
Tandem mCherry-EGFP-LC3B-expressing cells were treated for 2 days with 1 μM ATRA. Data were acquired on a FACS LSR-II (BD) using BD FACSDiva software and analyzed with FlowJo software. A gate was used based on parental cells to estimate the percentage of high autophagic activity as previously described [49 (link)].
For annexin V staining, 0.5 × 10⁶ cells were washed with cold PBS/5% BSA, resuspended in 70 μl binding buffer, and labeled with phycoerythrin- (PE-) labeled antibody against annexin V according to the manufacturer's protocol (BioVision). Caspase 3/7 activation was measured using Caspase-Glo™ 3/7 Assay according to the manufacturer's protocol (Promega Corporation, Madison, USA).

HEK293T cells were transfected with genes encoding human WT SMO tagged with Myc-DDK or human SMO D473H mutant tagged with Myc. Cells were washed in PBS supplemented with 0.5% FBS, fixed in 4% paraformaldehyde in PBS for 10 min, and incubated for 6 h at 37 °C in the same medium supplemented with BODIPY-cyclopamine (5 nM), with and without 4s at different concentrations (1 nm at 5 µM). The cells were permeabilised with 0.2% Triton X-100 (Sigma, St. Louis, MO, USA). Dako Fluorescent Mounting solution (Dako, Carpinteria, CA, USA) was used as mounting medium and the Hoechst reagent for staining of cell nuclei. BODIPY (green) and Hoechst (blue) signals were analysed in three representative fields per coverslip (×20 magnification). Data were expressed as the percentage of BC incorporation observed with BC alone.

PANC-1 cells were treated with Crizotinib (1 μM and 10 μM) for 6 hr and then added with a mitochondrion-specific dye (MitoTracker red FM: Molecular probes Inc, Eugene, OR) for another 30 min at 37°C. The cells were washed with PBS and fixed in an acetone: methanol solution (1:2) for 10 min at −20°C. After washing with PBS for several times, the cells were incubated with cytochrome c antibody (1:50; Santa Cruz Biotechnologies, Santa Cruz, CA) overnight for 4°C. On the following day, the cells were again washed with PBS several times and incubated with a mouse fluorescence-labeled secondary antibody (1:100, Dianova, Hamburg, Germany) for 1 h at room temperature. The cells were stained with DAPI to visualize the nuclei. Finally, the cells were covered with a fluorescent mounting solution (Dako, Carpinteria, CA) before viewing with a confocal laser scanning microscope (Olympus).