3D DNA FISH: A Technique to Locate a Specific Gene on a Chromosome

Abstract

Source: Gallerani, G. et.al. Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH. J. Vis. Exp. (2017).

Circulating tumor cells (CTC) isolation and characterization from the blood of cancer patients is a type of non-invasive liquid biopsy and associated with relapse and disease progression. This video describes the technique of DNA fluorescence-in-situ-hybridization (DNA FISH) on CTCs captured on a medical wire, to study a specific gene of interest.

Protocol

1. 3D DNA-FISH Day 2

CAUTION: Special attention must be paid to the high temperature of the instruments and humid chamber.

1. Set up the hybridization oven and dry heat oven (~ 3 h before starting the DNA-FISH protocol). Set the hybridization oven at 75 °C, set the dry heat oven at 37 °C, and cool the 0.4× SSC solution (pH = 7.0 ± 0.1) to 4 °C.
   NOTE: The pH status of FISH buffers is critical and must be 7.0 ± 0.1.
2. Wash the wire 3 times in ice-cold 0.4× SSC solution.
   NOTE: Keep the wire in the dark to avoid ....

Results

Figure 1: Functionalized wire, schematic arrangement of ALK break-apart probes, scheme of expected patterns of ALK rearrangement, and special holder. (a) Red boxes highlight the three main parts of the wire: functional.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tween 20 	BIO RAD	 # 1706531
Vysis 20X SSC (66g) 	Abbott Molecular Inc.	 # 30-804850	 powder to be resuspended in distilled water, as recommended
Water	MilliPore
DAPI, FluoroPure grade	 Invitrogen	# D21490
ZEISS Fluorescent Microscope Axioskop 	ZEISS