Characterization of Small Bowel Neuroendocrine Tumor Spheroids Using Immunofluorescence

Abstract

Source: Ear, P. H., et. al. Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids. J. Vis. Exp. (2019)

This video demonstrates the immunofluorescence staining of small bowel neuroendocrine tumor spheroids. The spheroids are fixed, permeabilized, and blocked and are then incubated with primary and secondary antibodies. A dye is added to stain nuclei before mounting and visualizing them under a fluorescent microscope.

Protocol

1. Small bowel neuroendocrine tumor (SBNET) collection and cell dissociation

1. Obtain resected patient SBNET samples after tumor tissue confirmation from the Surgical Pathology Core.
2. Cut SBNETs into 5 mm cubes and store them in 25 mL of Dulbecco's Modified Eagle Medium (DMEM)/F12 medium in a conical tube for transportation to the laboratory.
3. Transfer the tumors to DMEM containing 1% fetal bovine ser....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-rabbit FITC	Jackson ImmunoResearch	11-095-152	Secondary antibody couple to a green fluorophore
Autostainer Link 48	Agilent Dako	Not Available	Automated system for antibody staining
Cell freezing container	Thermo Scientific	5100-0001	Container to for freezing cells
CellSence	Olympus	Version 1.18	Computer software for using fluorescent microscope
Chromogranin A antibody	Abcam-45179	RB-9003-PO	Antibodies for IF
Collagenase	Sigma	C0130	Enzyme for digesting tumor tissue
DMEM	Gibco	11965-092	Medium for tissue preparation
DMEM/F12	Gibco	11320-033	Medium for organoid cultures
DNAse	Sigma	DN25	Enzyme for digesting tumor tissue
FBS	Gibco	16000044	Reagent for culture media
Fluorescent microscope	Olympus	CKX35	Microscope for taking pictures of SBENT spheroids
Glutamine	Gibco	A2916801	Reagent for culture media
Insulin	Sigma	I0516	Reagent for culture media
Matrigel	Corning	356235	Matrix to embed and anchore organoids
Mounting medium (VECTASHIELD)	Vector Laboratories	H-1200	Fixative for labelled-cells with a nuclear stain
Nicotinamide	Sigma	72340	Reagent for culture media
Paraformaldehyde	Electron Microscopy Sciences	15710	Reagent to fix cells
PEN/STREP	Gibco	15140-122	Reagent for culture media
SSTR2 antibody	GeneScritp	A01591	Antibodies for IF
Synaptophysin antibody	Abcam	32127	Antibodies for IF
TritonX	Mallinckrodt	3555 KBGE	Reagent to permeablize cells
Y-2763 ROCK inhibitor	Adipogen	AG-CR1-3564-M005	To improve SBNET spheroid viability after freeze thaw