Murine Intestinal Organoid Staining

Murine intestines were prepared as described previously^{5 (link)}. In situ hybridisation (ISH) assay was carried out as described previously^{5 (link)}. Organoids were immuno-stained either as whole-mount samples or as paraffin sections. Samples for IHC were processed as outlined before^{5 (link),24} and the following primary antibodies were used: ZEB2/SIP1 (H260; Santa-Cruz, or from Dr. Tulchinsky), E-cadherin (610181; BD), Vimentin (RV202; Santa-Cruz), α-SMA (ab5694; Abcam), Ki-67 (M7249; Dako) and Mucin2 (H300; Santa-Cruz). For IF, samples were exposed to goat anti-rabbit antibodies conjugated to Alexa Fluor594 (A11037; Invitrogen) and/or rabbit anti-mouse antibodies conjugated to Alexa Fluor488 (A11059; Invitrogen). Tetramethylrhodamine-B isothiocyanate (TRITC)-conjugated phalloidin (P1951; Sigma) was used to label actin filaments according to the manufacturer’s instruction.

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Li N., Babaei-Jadidi R., Lorenzi F., Spencer-Dene B., Clarke P., Domingo E., Tulchinsky E., Vries R.G., Kerr D., Pan Y., He Y., Bates D.O., Tomlinson I., Clevers H, & Nateri A.S. (2019). An FBXW7-ZEB2 axis links EMT and tumour microenvironment to promote colorectal cancer stem cells and chemoresistance. Oncogenesis, 8(3), 13.

Publication 2019

ZEB2/SIP1 E-cadherin Vimentin α-SMA Mucin2 Alexa Fluor594 A11037 Alexa Fluor488 A11059

Actin filaments Anti antibodies Antibodies Assay E cadherin Goat In situ hybridisation Intestines Isothiocyanate Mouse Mucin2 Murine Organoids Paraffin Phalloidin Rabbit Tetramethylrhodamine isothiocyanate Tetramethylrhodamine phalloidin Vimentin

Corresponding Organization :

Other organizations : University of Nottingham, Wellcome Centre for Human Genetics, University of Leicester, Hubrecht Institute for Developmental Biology and Stem Cell Research, John Radcliffe Hospital, The Seventh Affiliated Hospital of Sun Yat-sen University, Sun Yat-sen University

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Variable analysis

independent variables

Preparation of murine intestines
In situ hybridisation (ISH) assay
Immunostaining of organoids as whole-mount samples or paraffin sections
Primary antibodies used: ZEB2/SIP1, E-cadherin, Vimentin, α-SMA, Ki-67, Mucin2
Fluorescent secondary antibodies used
TRITC-conjugated phalloidin used to label actin filaments

dependent variables

Expression patterns and localization of proteins detected by immunostaining

control variables

Protocols followed as described previously in reference 5 (https://pubmed.ncbi.nlm.nih.gov/21282377/)
Samples for IHC processed as outlined before in references 5 (https://pubmed.ncbi.nlm.nih.gov/21282377/) and 24 (no link found)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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