3D DNA FISH: A Technique to Locate a Specific Gene on a Chromosome
Published: April 30, 2023
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. 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…
Representative 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: functionalized tip, wire-stopper, and un-functionalized tip. The functionalized tip is the most delicate part of the wire and must not be touched …
Açıklamalar
The authors have nothing to disclose.
Materials
| 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 |
Bu Makaleden Alıntı Yapın
3D DNA FISH: A Technique to Locate a Specific Gene on a Chromosome. J. Vis. Exp. (Pending Publication), e20252, doi: (2023).