Ultracruz mounting medium with dapi

Paraformaldehyde-fixed and paraffin-embedded tissues were sectioned (4 μm) and mounted on chromogelatin-precoated slides (Menzel-Glaser). Sections were deparaffinized, rehydrated, and blocked using 10% normal donkey serum (Jackson Immunoresearch). Next, slides were incubated with primary mouse monoclonal anti-CD63 antibody (Abcam, cat. # ab8219, 1:30) overnight at 4 °C. Secondary antibody, Cy3-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, cat. # 715-165-150, 1:1 000) was applied and incubated for 1 hr at room temperature. Negative controls were performed without primary antibodies. Finally, sections were mounted in Ultra Cruz Mounting Medium with DAPI (Santa Cruz Biotechnology) and visualized with an epifluorescent microscope Zeiss Axio Imager System (Carl Zeiss).

For light microscopy, tissues and organoids were xed for 16 h in 10% neutral buffered formalin at 4 °C.
The tissues were embedded in para n and cut into 5-μm-thick sections. Sections were stained with hematoxylin and eosin (H&E) or subjected to immunohistochemistry. Organoids were embedded in Tissue-Tek ® O.C.T. Compound (Sakura Finetek), frozen at -20 °C, and cut into 10-µm-thick sections. For immunohistochemistry, para n sections were prepared by boiling in 10 mM citrate buffer pH 6.0 for 15 min. After blocking with 1% bovine serum albumin, sections were incubated for 16 h at 4 °C with the following primary antibodies: anti-lysozyme C (1:200, goat polyclonal, Santa Cruz Biotechnology); antiproliferating cell nuclear antigen (PCNA, 1:100, mouse monoclonal, Dako); anti-5-mC (1:300, rabbit monoclonal, Cell Signaling Technology); and anti-H3K4, 9, 27, 36, and 79me3 (1:300, rabbit monoclonal, Cell Signaling Technology). Before incubation with secondary antibodies, the tissues were washed to remove the unbound antibodies. The sections were then incubated with secondary antibodies (1:1000 dilution; Alexa Fluor 488 and/or Alexa Fluor 555, Invitrogen) for 1 h at 25 °C and mounted using UltraCruz™ Mounting Medium with DAPI (Santa Cruz Biotechnology). The samples were observed under a uorescence microscope (Axioskop 2 plus or Apotome. 2, Carl Zeiss).

The primary antibody of FLT3 (Santa Cruz) and αSMA (Abcam) was applied to the cryo-sections (5 μm) of tumor xenografts at a 1:200 concentration for 1 h at room temperature. FITC-tagged anti-rabbit IgG secondary antibody (Sigma-Aldrich) (1:200) was used for 1 h at room temperature. Slides were mounted with UltraCruz mounting medium with DAPI (Santa Cruz) for counter staining and observed with a fluorescent microscope (Leica TCS/SP5). The excitation wavelengths for FITC and DAPI were 490 nm and 359 nm, respectively.

Cells were grown on coverslips and following methanol fixation, blocking was performed at room temperature for 1 h. Anti-FLT3 antibody (Santa Cruz; 1:200) incubation was done at room temperature for 1 h. Alexa Fluor 568-tagged anti-rabbit IgG secondary antibody (Invitrogen, USA) (1:1000) was used for another 1 h at room temperature. Coverslips were mounted with UltraCruz mounting medium with DAPI (Santa Cruz) for counter staining. Slides were observed with a fluorescent microscope (Leica TCS/SP5). The excitation wavelengths for Alexa Fluor 568 and DAPI were 578 nm and 359 nm, respectively.