Lysozyme

Organoids immunocytochemistry had been developed previously [15 (link)]. Samples were thoroughly covered with primary antibodies at 4°C overnight. Antibodies used for the staining were as follows: mucin 2 (Santa Cruz Biotechnology, Santa Cruz, CA), Ki67 antibody (Abcam, Cambridge, MA), chromogranin A (Santa Cruz), lysozyme (Diagnostic BioSystems, Fremont, CA), mucin 5AC (Santa Cruz Biotechnology, Santa Cruz, CA), and villin (Santa Cruz Biotechnology, Santa Cruz, CA) The next day, the samples were embedded with secondary antibodies at room temperature for 2 h, and Alexa Fluor 594 conjugated anti-rabbit IgG and Alexa Fluor 594 conjugated anti-mouse IgG (Life Technologies, Gaithersburg, MD).

Culture medium was removed, and the organoids in Matrigel were washed with DPBS. Fixation was done with 4% Paraformaldehyde until the Matrigel was disintegrated. Permeabilization was performed with 0.1% Tween-20 and 0.2% Triton-X100 in PBS buffer, and then, 5% BSA in DPBS was used to reduce non-specific binding. Samples were covered with primary antibodies at 4 °C overnight. The next day, the samples were embedded with secondary antibodies at room temperature for 2 h. Antibodies used for the staining were as follows: Mucin 2 (Santa Cruz Biotechnology, Santa Cruz, CA), Ki67 antibody (Abcam, Cambridge, MA), Lgr5 (Abgent, San Diego, CA), E-cadherin (Santa Cruz Biotechnology), ChromograninA (Santa Cruz), β –catenin (Abcam), Lysozyme (Diagnostic Biosystems, Fremont, CA), α-SMA (Sigma-Aldrich), Vimentin (BD Biosciences), and Alexa Flour 488 conjugated anti-rabbit IgG and Alexa Flour 594 conjugated anti-mouse IgG (Life Technologies, Gaithersburg, MD).