Rabbit anti epcam

For whole-mount staining, the organoids were first fixed using 4% paraformaldehyde (Thermo Fisher) for 30 min at room temperature and then permeabilized using 0.1% TritonX-100 (Sigma-Aldrich) in Tris-buffered saline (TBS) for 15 min. After blocking in DAKO (Agilent) for 1 h, organoids were incubated with primary antibodies overnight at 4°C. Next day, after triple washing, organoids were incubated in secondary antibodies (ThermoFisher) along with 4’, 6-diamidino-2-phenylindole (DAPI) for 1 h at room temp. The following primary antibodies were used: mouse anti-vimentin (Abcam), rabbit anti-calcitonin gene related peptide (CGRP) (Millipore Sigma), rabbit anti-EpCAM (Abcam), rabbit anti-synaptophysin (Abcam), mouse anti-chromogranin A (Proteintech), NCAM1 (Cell Signaling Technology). Confocal imaging was performed using Zeiss LSM 880.

Human bronchial epithelial (16HBE) cells were obtained from Shanghai Institute for Biological Science. Cells were cultured in RPMI 1640 (HyClone, UT, USA) supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (BIO International, Auckland, New Zealand). All cells were maintained at 37 °C in a humidified incubator with 5% carbon dioxide. Human recombinant CCN1 proteins were obtained from PeproTech (Shanghai, China). The rabbit anti-EpCAM as well as rabbit anti-CD31 were purchased from Abcam (HK, China). Mouse anti-CCN1 and horseradish peroxidase-coupled secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

The double immunofluorescence antigen labelling of frozen human lung sections was performed with LeA, LeX, SLeA and SLeX, with EpCAM antibody. The frozen human lung sections were fixed in Acetone/Ethanol (75:25) for 10 min and washed twice with TBS. The sections were blocked with 5% normal donkey serum (Jackson ImmunoResearch Lab Inc, West Grove PA) for an hour at room temperature. The sections were incubated with rabbit anti-EpCAM (1:300, Abcam, Cat#: ab32392) plus (1) FITC conjugated mouse anti-LeA (1:100, Santa Cruz, cat#: sc-51512) and (2) FITC conjugated mouse anti-human CD15 (LeX, 1:50); (3) mouse anti-SLeA (1:500, CA19.9) and (4) Rat anti-SLeX (1:200, BD 555946) overnight at 4 °C. The slides were washed three times in TBS and incubated with Alexa 647 conjugated donkey anti-rabbit secondary antibodies (Jackson ImmunoResearch Lab, 1:300) for (1) and (2) and CY3 conjugated donkey anti-mouse (Jackson ImmunoResearch Lab, 1:300) and Alexa 647 conjugated donkey anti-rabbit secondary antibodies (Jackson ImmunoResearch Lab, 1:300) for (3) and CY3 conjugated donkey anti-rat (Jackson ImmunoResearch Lab, 1:300) and Alexa 647 conjugated donkey anti-rabbit secondary antibodies (Jackson ImmunoResearch Lab, 1:300) for (4). Samples were then washed three times with TBS and mounted with Prolong Gold anti-fade mounting media containing DAPI (Invitrogen).