Ab17133

5 μm thick organoid sections were created from paraffin-embedded constructs, and then deparaffinized for staining. IHC was used to visualize biomarkers cytokeratin 5/6 (CK5/6), calretinin, and thrombomodulin. Blocking was performed by incubation under Dako Protein Block for 15 minutes. Primary antibodies CK5/6 (Abcam, ab17133, raised in mouse) and calretinin (Abcam, ab702, raised in rabbit) or CK5/6 and thrombomodulin (Abcam, ab109189, raised in rabbit) were applied to the sections on the slides at a 1:200 dilution in Dako Antibody Diluent and incubated at room temperature for 1 hour. Next, secondary Alexa Fluor 488 or Alexa Fluor 594 antibodies with appropriate species reactivity were applied to all samples at 1:200 in Dako Antibody Diluent and left at room temperature for 1 hour (anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 594, Life Technologies, Carlsbad, CA, A-11070). Sections were then incubated with Dapi for 5 minutes prior to coverslipping. For Annexin V and Ki67 staining (Abcam, Cambridge, MA, ab14196 and ab16667, respectively) in subsequent biomarker-driven experiments, an identical protocol was employed. Fluorescence images were taken using a Leica DM400B Compound Microscope and overlaid for analysis.

Tumor tissues and organoids were fixed in 10% neutral buffered formalin followed by gradient dehydration, wax immersion, paraffin embedding, and sectioning. For processing organoids, prestaining of eosin was performed during dehydration in order to locate organoids in paraffin blocks and sections. Haematoxylin–eosin (HE) staining and immunostaining was performed using standard protocols on 5 μm sections^{72 (link),73 (link)}. The antibodies used were listed as following: AE1/3 (1:50, Abcam, ab27988), Vimentin (1:100, CST, 5741 S; 1:100, Santa Cruz, sc-6260), CK5/6 (1:50, Abcam, ab17133), p63 (1:50, Abcam, ab124762), MST1R (1:100, ATLAS, HPA008180), LMP1 (1:100, Abcam, ab78113), Ki67 (1:400, CST, 9449 S), and CD3e (1:100, Dako, A0452).