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OPEN ACCESSProtocolDetection of chromosome instability by
interphase FISH in mouse and human tissuesRaul Torres-Ruiz,
Tatiana P. Grazioso,
Marta Brandt, Marta
Martinez-Lage,
Sandra
Rodriguez-Perales,
Nabil Djouder
rtorresr@cnio.es (R.T.-R.)
srodriguezp@cnio.es
(S.R.-P.)
ndjouder@cnio.es (N.D.)
Highlights
A protocol for single-
cell CIN analysis in
paraffin-embedded
tissues
Design of FISH
probes for CIN
detection
Identification,
visualization, and
quantification of CIN
by FISHChromosomal instability (CIN), a type of genomic instability, favors changes in chromosome
number and structure and it is associated with the progression and initiation of multiple diseases,
including cancer. Therefore, CIN identification and analysis represents a useful tool for cancer
diagnosis and treatment. Here, we report an optimizedmolecular-cytogenetic protocol to detect
CIN in formalin-fixed, paraffin-embedded mouse and human tissues, using fluorescent in situ
hybridization to visualize and quantify chromosomal alterations such as amplifications, deletions,
and translocations.Torres-Ruiz et al., STAR
Protocols 2, 100631
September 17, 2021 ª 2021
The Author(s).
https://doi.org/10.1016/
j.xpro.2021.100631
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OPEN ACCESSProtocolDetection of chromosome instability by interphase FISH
in mouse and human tissues
Raul Torres-Ruiz,1,4,* Tatiana P. Grazioso,2,4 Marta Brandt,2,3 Marta Martinez-Lage,1
Sandra Rodriguez-Perales,1,5,* and Nabil Djouder2,5,6,*1Molecular Cytogenetics and Genome Editing Unit, Human Cancer Genetics Program, Centro Nacional de Investigaciones
Oncolo´gicas (CNIO), 28029, Madrid, Spain
2Molecular Oncology Programme, Growth Factors, Nutrients and Cancer Group, Centro Nacional Investigaciones
Oncolo´gicas (CNIO), Madrid, 28029, Spain
3Present address: Broad Institute of MIT and Harvard, Cambridge, MA, USA
4These authors contributed equally
5Technical contact
6Lead contact
*Correspondence: rtorresr@cnio.es (R.T.-R.), srodriguezp@cnio.es (S.R.-P.), ndjouder@cnio.es (N.D.)
https://doi.org/10.1016/j.xpro.2021.100631SUMMARY
Chromosomal instability (CIN), a type of genomic instability, favors changes in
chromosome number and structure and it is associated with the progression
and initiation of multiple diseases, including cancer. Therefore, CIN identification
and analysis represents a useful tool for cancer diagnosis and treatment. Here,
we report an optimized molecular cytogenetic protocol to detect CIN in
formalin-fixed, paraffin-embedded mouse and human tissues, using fluorescent
in situ hybridization to visualize and quantify chromosomal alterations such as
amplifications, deletions, and translocations.
For complete information on the generation and use of this protocol, please refer
to Brandt et al. (2018).BEFORE YOU BEGIN
Fluorescence in situ hybridization (FISH) analysis
FISH probes are short fluorescently-labeled DNA fragments complementary to a specific region of
interest, and produced from template plasmids that contain large genomic inserts. Though, there
are plenty of commercially available FISH probes used for human sample analysis, the list of FISH
probes for mouse samples is very restricted, making it necessary to design and prepare new custom-
ized probes.
Probes can be produced using various cloning vectors such as: cosmids, fosmids, PI bacterial artifi-
cial chromosomes (BACs) and filamentous phage artificial chromosomes (PACs), wich major differ-
ence is the length of the probe. Cosmid and fosmids have a size of 
40 kb, BACs size ranges be-
tween 
150–350 kb and the size of PACs is 
300 kb. However, the most commonly employed
are BACs and PACs since they yield brighter FISH signals. Importantly, probes smaller than cosmids
are associated with weak fluorescence.
Clones can be located using:
 UCSC Human Genome Browser: (http://genome.ucsc.edu)
 NCBI Clone Finder database (http://www.ncbi.nlm.nih.gov/mapview/mvhome/clonehome.html)STAR Protocols 2, 100631, September 17, 2021 ª 2021 The Author(s).
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1
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OPEN ACCESS ProtocolMoreover, clones can be obtained from:
 BACPAC Resources Center (https://bacpacresources.org/)
 Source Bioscience (https://www.sourcebioscience.com/life-science-research/clones/genomic-clones/).
The protocol bellow describes the specific steps to find BACs for a particular locus using the UCSC
Human Genome Browser, and thenafter, FISH-probe design.BAC clone selection
Timing: 1 h
1. Go to Genome Browser webpage (https://genome.ucsc.edu/cgi-bin/hgGateway) and select
mouse as a model organism (top left mouse brown icon).
2. Chose a region of interest by typing a gene name or the locus position in the search window.
3. In order to visualize BACs information, select the BACs libraries of choice in the Clone Ends Track
Setting window and select clone ends (Figure 1A).
4. Make your BAC selection (Figure 1B).
a. Order the selected BAC clones.KEY RESOURCES TABLEREAGENT or RESOURCE SOURCE IDENTIFIER
Bacterial and virus strains
Bacterial cells from E. coli BACPAC Resources Center
Chemicals, peptides, and recombinant proteins
Glycerol Sigma G-6279
Glucose Sigma G8769
Tris Sigma T1503
EDTA Merck 1084520250
NaOH Merck 106462
SDS Bio-Rad 1610302
Potassium acetate Sigma P1190
Isopropanol Sigma I9516
Ethanol Sigma 51976
RNAse QIAGEN 19101
Sodium acetate Sigma S7899
Glacial acetic acid Sigma A6283
Agarose Conda 8010.22
Xylene AnalaR NORMAPURE 28975.291
Green-dUTP Abbott Molecular 02N32-050
Red-dUTP Abbott Molecular 02N34-050
HCL Fisher Chemical 10467640
PBS Sigma D8537
Wash Buffer Agilent Technologies G9401A;G9402A
Proteinase K Dako S3020
Vysis LSI/WCP hybridization buffer Vysis 30-804826
VECTASHIELD Antifade Mounting Medium with DAPI Vector Laboratories H-1200
VECTASHIELD Antifade Mounting Medium Vector Laboratories H-1400
Buffer TE Invitrogen AM9849
Sspe 203 buffer Gibco 15591-043
(Continued on next page)
2 STAR Protocols 2, 100631, September 17, 2021
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
N-Lauroylsarcosine sodium salt solution Sigma L-7414
Sodium borohydride Sigma 452882
BSA Sigma A9418
Post-Hybridization Wash Buffer Vitro-Master diagnostica MAD-FS0105-1
Critical commercial assays
Nick Translation Kit Roche 10976776001
SureTag DNA Labeling Kit Agilent Technologies 5190-3399
SureTag DNA Labeling Kit Purification Columns Agilent Technologies 5190-3391
Dako’s Histology FISH Accessory Kit Dako K5799
Vysis IntelliFISH Universal FFPE Tissue
Pretreatment and Wash Reagents
Vysis 08N85-005
Vysis IntelliFISH Hybridization Buffer Vysis 08N87-001
DNeasy Blood & Tissue Kits QIAGEN Cat#69506
Oligo aCGH/Chip-on-chip Hybridization Kit Agilent Technologies 5188-5220
SurePrint G3 Mouse Genome CGH
Microarray Kit, 43180K
Agilent Technologies G4839A
SurePrint G3 Human CGH Microarray Kit, 43180K Agilent Technologies G4449A
SurePrint G3 Human CGH Microarray Kit, 8360K Agilent Technologies G4450A
Pretreatment Kit Vitro-Master diagnostica MAD-FISH-PTK
Experimental models: organisms/strains
Mouse: Mouse: MCRS1lox/lox: Mcrs1tm1.1Ndj Dr. Nabil Djouder, (Fawal
et al., 2015) Spanish National
Cancer Research Center (CNIO)
J:232965
Mouse: mTORlox/lox: B6.129S4-Mtor tm1.2Koz/J (Risson et al., 2009) J:011009
Mouse: p53 lox/lox: B6.129P2-Trp53tm1Brn/J (Marino et al., 2000) J:61961
Mouse: Villin-CreERT2 : Tg(Vil-cre/ERT2)23Syr (el Marjou et al., 2004) J:92295
Software and algorithms
CytoVision Leica Microsystems N/A
Other
Mouse Cot-1 DNA Invitrogen Cat#18440-016
Human Cot-1 DNA Invitrogen Cat#15279-011
Positively charged glass slides Menzel-Glaser N/A
ThermoBrite instrument Leica N/A
Leica DM5500B fluorescence microscope Leica N/A
Rubber cement Marabu 29010010000
25350 mm Cover slips Menzel-Glaser 15747592
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OPEN ACCESSProtocolMATERIALS AND EQUIPMENT
GTE buffer
The GTE buffer contains 50 mM Glucose, 25 mM TrisHCI pH 8.0, and 10 mM EDTA pH 8.0; It has to
be filter sterilized.
Storage conditions: It can be kept at 4C for several days.Reagent Final concentration Amount
Glucose 1 M 50 mM 5 mL
TrisHCl 1 M pH 8.0 25 mM 2.5 mL
EDTA 0.5 M pH 8.0 10 mM 2 mL
Milli-Q Water n/a 90.5 mL
Total n/a 100 mL
STAR Protocols 2, 100631, September 17, 2021 3
Figure 1. BAC clone selection for the preparation of FISH probes using the UCSD genome browser
Screenshot of the Genome Browser Display.
(A) (1). Once you have selected your region of interest you need to adjust tracks: to visualize BACs information make sure that you have activated the
‘‘clone ends’’ display option to ‘‘Full’’. Information from different BAC libraries can be activated from the clone ends track settings. (2). Different
companies provide clones from different libraries.
(B) You can change the display: zoom in or zoom out, display additional genomic features, etc. (3). Select the appropriate item from search results.
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OPEN ACCESS ProtocolLysis solution
The lysis solution contains 200 mM NaOH, 1% SDS buffer; It has to be filter sterilized. Storage con-
ditions: It can be kept between 20C–25C until its use.4 STAR Protocols 2, 100631, September 17, 2021
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OPEN ACCESSProtocolNote: The lysis solution has to be made freshly every time that it would be necessary.Reagent Final concentration Amount
NaOH 10 M 200 mM 0.2 mL
20% SDS buffer 1% (w/v) 0.5 mL
Milli-Q Water n/a 9.3 mL
Total n/a 10 mLNeutralization solution
The neutralization solution contains 7.5 M Potassium acetate at pH 5.5.
Storage conditions: It can be stored at 4C for several weeks.Reagent Final concentration Amount
Potassium Acetate 5 M 60 mL
Glacial acetic acid n/a 11.5 mL
Milli-Q Water n/a 28.5 mL
Total n/a 100 mLNick translation mix
The Nick Translation mix should be prepared on ice and with reduced light exposure; the enzyme
should be the last component added to the mix.
Note: The Nick Translation mix has to be prepared freshly every time it would be needed.Reagent Final concentration Amount
103 Nick Translation buffer 13 5 uL
0.1 mM dNTP mix (dATP/dGTP/dCTP) 0.02 mM 10 uL
0.1 mM dTTP 0.01 mM 5 uL
0.1 mM Fluorescence labeled dUTP 0.01 mM 5 uL
Extracted DNA 20 ug/mL 1 ug
Nick Tranlation enzyme (DNAse I) n/a 10 uL
Nuclease-free water n/a Up to 50 uL
Total n/a 50 uLSTEP-BY-STEP METHOD DETAILS
BAC DNA isolation
Timing: 3 h and two incubations of 8–16 h
Note: Stab cultures are typically shipped at 20C–25C and should be placed at 4C upon
arrival. The culture has few weeks of finite life at 4C, thus, a glycerol stock should be prepared
for long term storage (described below). The addition of glycerol stabilizes the frozen bacte-
ria, preventing cell membrane damage keeping the cells alive. A glycerol stock of bacteria can
be stored stably at 80C for many years. To recover the bacteria from the glycerol stock,
open the tube and use a sterile loop to scrape the frozen bacteria off from the top, andSTAR Protocols 2, 100631, September 17, 2021 5
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OPEN ACCESS Protocolgrow the bacteria from 12–16 h using the appropriate medium. Importantly, do not let the
glycerol stock unthaw.
The protocol below describes a rapid alkaline lysis miniprep method. The method works very well
producing high quality DNA in terms of concentration and purity. However, commercial alternatives
such as Nucleobond Xtra BAC kit (Macherey-Nagel), BACMAX purification kit (Cambio) or QIAGEN
Large-construct kit (QIAGEN) are also available.
1. Streak a small amount of E. coli onto a selective plate and incubate from 12–16 h at 37C to obtain
single colonies.
2. Pick a single colony and inoculate into 10 mL of lysogeny broth (LB) medium containing the se-
lective antibiotic in a 50 mL falcon tube.
CRITICAL: Culture poor aeration may contribute to produce a low DNA yield. The optimalculture volume to air volume ratio is 1:4 or less.3. Grow with vigorous shaking at 37C in a shaking incubator for 12–16 h.
Note: Freeze each plasmid mixing 800 ml of bacteria culture and 200 ml of sterile glycerol.
Store at 80C.
4. Harvest the bacterial cells by centrifuging at 1792 g (4,000 rpm) for 10 min. The temperature or
the spin is not critical at this stage. Carefully discard the supernatant.
5. Resuspend the bacterial pellet in 300 mL of GTE buffer and transfer it to a 2.2 mL centrifuge tube.
6. Add 600 mL of freshly made lysis solution, mix gently by inverting, and incubate on ice for 5 min.
CRITICAL: From this step onwards, the DNA is already suspended in the solution,improper handling by incomplete mixing of buffer, excessive mechanical treatment or
by prolonged incubation during the lysis step may contribute to genomic DNA contam-
ination or DNA shearing, thus, avoid vigorous shaking and repetitive pipetting. Mix
gently by inverting the tube four to six times.7. Slowly add 500 mL of neutralization buffer drop by drop, mix by inverting the tube four to six times
(do not vortex), and incubate on ice for 10 min. A thick white precipitate of bacterial DNA and
protein will form.
a. Centrifuge at 11200 g (10,000 rpm) for 10 min at 4C.
8. Transfer the supernatant using a P1000 pipette to a new 2.2 mL centrifuge tube.
a. Centrifuge at 11200 g (10,000 rpm) for 5 min at 4C.
9. Transfer the supernatant using a P1000 pipette to a new 1.5 mL centrifuge tube containing 700 mL
of ice-cold isopropanol trying to avoid any white precipitate material.
a. Mix by inverting the tube a few times.
b. Centrifuge at 21952 g (14,000 rpm) for 20 min at 4C.
10. Discard the supernatant and wash the DNA pellet with 0.5 mL of 70% ethanol.6a. Mix by inverting the tube several times
b. Centrifuge at 21,952 g (14,000 rpm) for 5 min at 4C.
11. Remove as much supernatant as possible with a pipette and air-dry the pellet at 20C–25C for
10 min.
12. Once the pellet turns from white to translucent in appearance, dissolve the DNA into 100 mL of
nuclease-free water by gentle occasional tapping of the tube.
13. Add 1 mL of RNAse (100 ug/mL) and incubate for 30 min at 37C.
14. Precipitate the DNA in a mix of 1/10 vol 3 M sodium acetate and 2.5 vol of 100% ethanol for
60 min at 80C.
a. Centrifuge at 21,952 g (14,000 rpm) for 15 min at 4C.STAR Protocols 2, 100631, September 17, 2021
Figure 2. Exemplary analytical check of DNA integrity after miniprep BAC clones purification
(A) Representative images of 5 ul of each precipitated BAC analyzed on a 1% agarose gel. The red square represents
the sheared and DNA smear low yield DNA extractions. (B) Suitable DNA for labeling protocol. Arrows indicate not
sheared BAC DNA.
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OPEN ACCESSProtocol15. Discard the supernatant and wash the DNA pellet with 0.5 mL of 70% ethanola. Centrifuge at 21,952 g (14,000 rpm) for 5 min at 4C.
16. Discard the supernatant and dissolve the DNA into 100 mL of nuclease-free water.
17. Check the DNA integrity by running a 1% agarose gel (Figure 2).
Note: The agarose gel electrophoresis allows us to confirm the efficiency of the Nick transla-
tion reaction by analyzing the product size distribution.
Note: The quality of the probe is vital for a successful FISH, and this is in turn strictly related to
the quality of the DNA template. Make sure to use good quality, purified DNA, free of RNA,
protein or other contaminants. The purity of the preparation should be determined using clas-
sical gel electrophoresis.
18. Store at 20C.
Note: The average yield of the DNA should be approximately 4–8 mg. Commercial alkaline
lysis/column purifications kits can be used as an alternative. However, in our hands the alkaline
lysis miniprep method described above produces high yields of high-quality DNA needed for
the Nick translation protocol.
CRITICAL: Multiple freeze-thraw cycles or storage at 4C during extended periods (1–
2 months) may produce extensive nicks and breakages. It is important to use freshly pre-
pared BACs, or store the DNA in alcohol in precipitated form, for a maximum of 4–
6 months.Fish probe preparation: DNA labeling
Timing: 2–3 h and 8–16 h of incubation
Note: Probes can be labeled by direct incorporation of nucleotides conjugated to fluoro-
chromes (i.e., fluorescein iso-thiocyanate (FITC) or tetramethyl rhodamine). Generally,STAR Protocols 2, 100631, September 17, 2021 7
Figure 3. Exemplary Analytical Check of Probe Size Determination by 1% Agarose Electrophoresis
As the incubation time increases the size distribution shifts to smaller probe fragments ((A) 60 min digestion; (B)
120 min digestion). Image A represents an optimal digestion experiment whereas image B is over-digested. Red
squares indicate the bulk of the fragments.
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OPEN ACCESS Protocoldeoxyuridine 50-triphosphate (dUTP) is conjugated directly to the fluorochrome and used in
the place of dTTP in a labeling reaction. Labeling approximately 1 mg of extracted DNA is
enough for ten FISH experiments when using a commercially available kit for labeling DNA
with modified dNTPs (i.e., Roche or ThermoFisher Nick Translation Kit).
19. Set-up the Nick translation reaction in a final volume of 50 mL in a tube on ice.8a. Vortex and centrifuge the tube for 10 s.
b. Incubate the tube for 90 min at 16C.
20. Place the reaction on ice and determine the product size distribution by running 5 mL of the mix
in a 1% agarose gel.
CRITICAL: Successful Nick translation will result in a smear with the bulk of the fragmentsrunning between 150 bp and 700 bp (Figure 3). When labeled DNA fragments are too small
(>150 bp), they might not hybridize efficiently to DNA; hence, FISH signals would not be
visible. Incubation time will vary according to the amount and quality of BAC DNA and
the effectiveness of the Nick translation enzyme.Note: The structure and net charge of the different fluorophores affect the migration pattern
on the gel. The unincorporated dUTP appears on the gel as brighter areas. Unincorporated
Green_dUTP appears at the bottom of the run and Orange_dUTP appears around the 500
bp region overlapping with the probe DNA smear.
21. If the size of the bulk of the fragments is larger than 500 bp incubate themix for another 30min at
16C, if not, inactivate DNaseI by incubating the mix at 75C for 10 min.
22. Purify the DNA with a PCR purification kit (i.e., QIAGEN), and elute in 60 mL of nuclease-free wa-
ter.
23. Precipitate the DNA in a mix of 1/10 vol 3 M sodium acetate (pH 5.2), 2.5 vol of 100% ethanol,
and 1 mg of Cot-1 DNA.
Note: Mouse or human Cot-1 DNA is a mixture of highly repetitive DNA sequences, use to
block non-specific hybridization of FISH probes.
24. Incubate from 12-16 h at 20C.STAR Protocols 2, 100631, September 17, 2021
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OPEN ACCESSProtocola. Centrifuge at 21,952 g (14,000 rpm) for 15 min at 4C.
25. Wash pellet with 500 mL 70% ethanol and allow to air dry.
26. Resuspend the pellet in 10 mL of nuclease free-water.
27. Store at 20C.
Note: The DNA is stable in water for long periods (up to 1–2 years) if stored at20C until use.
Formalin-fixed paraffin-embedded (FFPE) tissue section preparation
Timing: 1 h to cut 5 blocks with 5 slides each
Note: Time dependents on the number of blocks and the number of cuts required.
In Brandt et al., FFPE of intestinal tissues obtained from MCRS1(lox/lox)Int, mTOR(lox/lox)Int and
p55(lox/lox)Int mice were used (Brandt et al., 2018). Importantly, these steps describe a general pro-
tocol for FFPE mouse and human tissue preparation. Thus any FFPE tissue derived from any organ-
ism (human/mouse/etc) can be used.
28. Cut FFPE tissue sections of 4–6 mm and mounted them on a poly-L-lysine-coated or positively-
charged glass slide.
Note: Use gloves at all times when handling specimens. All reagents should be handled as if
hazardous.
Pretreatment of FFPE tissue sections
Timing: 3 h and 8–16 h of incubation
Note: This step describes the protocol to pre-treat FFPE tissue sections. Formalin fixation in-
troduces a macromolecular network which significantly reduces the access of FISH probes to
target DNA. For that reason, the slides need to be pre-treated to diminish the extracellular
matrix of proteins which potentially limit the accessibility of the probe to the cells, preventing
tissue autofluorescence. Only tissues preserved in neutral-buffered formalin and paraffin-
embedded are suitable for use. Other fixatives are not suitable. The use of tissue exposed
to acid decalcification is not recommended. EDTA as decalcifier has been reported to pre-
serve the DNA better for FISH techniques.
29. Melt the paraffin by incubating the slides at 65C for 12–16 h.
30. Deparaffinize the tissue slides in a xylene bath and incubate for 3 min at 65C.
CRITICAL: Xylene can affect a person’s nervous, respiratory and cardiovascular system.When handling xylene, work in a well-ventilated area, preferably a fume hood with down-
draft fume extraction wearing protective clothing. Xylene must be properly stored in a
well-ventilated area, that maintains a consistent temperature (15C–25C).31. Change the xylene bath and repeat once.a. Tap gently on a paper towel to draw off the excess of liquid.32. Place the slides in 100% ethanol for 3 min at 20C–25C.
33. Change the ethanol bath and repeat once.a. Tap off the excess of liquid.34. Place the slides in 80% ethanol for 3 min at 20C–25C.
a. Tap off the excess of liquid.35. Place the slides in 70% ethanol for 3 min at 20C–25C.STAR Protocols 2, 100631, September 17, 2021 9
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OPEN ACCESS Protocol36. Heat the water bath and the Pre-Treatment (10 mM citrate buffer, pH 6.0) solution at 95C–99C
in a plastic Coplin jar.
Note: The Pre-Treatment Solution is designed for single use application only.
37. Immerse the deparaffinized sections into 0.2 N HCL for 20 min at 20C–25C.
38. Incubate the slides into the pre-heated Pre-Treatment solution for 20 min at 95C–99C.
39. Allow the slides to cool in the Pre-Treatment Solution for 15 min at 20C–25C.
40. Wash the slides in a Coplin jar with 13 PBS and soak the slides for 3 min at 20C–25C.10a. Replace 13 PBS and soak the sections for another 3 min.41. Dry the slides by touching gently the bottom edge and wiping the underside with a paper towel.
Alternatives: Commercially available kits include Dako’s Histology FISH Accessory Kit, or Vy-
sis IntelliFISH Universal FFPE Tissue Pretreatment andWash Reagents, and can be used for the
pre-treatment protocol.
CRITICAL: Breaking of peptide bonds by protease digestion is the key step of this proto-col, affecting directly the signal quality since it allows the access of the FISH probes to the
target DNA and reduces autofluorescence.42. Digest the pre-treated tissue by covering it with 40 mL of ready to use pepsin.a. Incubate samples for 30 min at 37C.
CRITICAL: Digestion for shorter time that needed can led to background autofluorescenceand incomplete hybridization; while over-digestion of tissue sections may result in the loss
of signal, loss of tissue morphology, and can cause sections to detach from the slides.43. Place the slides in 70% ethanol for 3 min at 20C–25C.
a. Change ethanol bath and repeat once.
b. Tap off the excess of liquid.44. Place the slides in 80% ethanol for 3 min at 20C–-25C.
a. Tap off the excess of liquid.45. Place the slides in 96% ethanol for 3 min at 20C–25C.
46. Air-dry the slides from 2 to 5 min.
47. Place the slide on a 45C–50C slide warmer to allow the remaining ethanol to evaporate.Probe hybridization
Timing: 0.5 h and 8–16 h of incubation
Note: This step describes the protocol to perform the FISH assay using a ThermoBrite instru-
ment that automates the denaturation and hybridization steps in the FISH protocol. Refer to
the ThermoBrite Operations Manual for instructions on instrument use.
Alternatives: A heated plate can be used for the denaturation steps, and a humidified cham-
ber or a plastic slide storage box containingmoist tissue paper, incubated at 37C can be used
for the hybridization steps.
48. Moisten humidity cards with water and place in the card slots of the ThermoBrite instrument.
Ensure that the surface of the ThermoBrite instrument is clean and free of debris.
49. Set the following conditions:STAR Protocols 2, 100631, September 17, 2021
Figure 4. Representative images of step-by-step coverslip mounting for FISH probe hybridization
From left to right, 10 mL of probe mixture is applied to the slide with the mounted tissue, immediately, an appropriated size coverslip is applied. Once no
bubbles are present, the coverslip is sealed with rubber cement. Ensuring a proper sealing is key to avoid evaporation. After hybridization using the
ThermoBrite denaturation-hybridization program (not shown), the rubber cement seal is carefully removed by pulling one of the corners of the seal with
forceps.
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OPEN ACCESSProtocola. Denaturation temperature (Melt Temp): 75C
Denaturation time (Melt Time): 3 min
b. Hybridization temperature (Hyb Temp): 37C
Hybridization time (Hyb Time): 16 h50. Thaw labeled probe and Vysis IntelliFISH hybridization buffer (08N87-001) at 20C–25C.
a. Prepare the probe mixture with 1 mL of labeled probe, 3 mL of nuclease-free water and 7 mL of
hybridization buffer.
b. Vortex the mix and centrifuge using a micro-centrifuge for 2–3 s at maximum speed.51. Apply 10 mL of the probe mixture to the center of each tissue section, and immediately apply a
coverslip. Ensure that no air bubbles are present in the probe mixture prior to applying the
coverslip (Figure 4).
Note: The volume of the probe mixture, and the size of the coverslip can be changed accord-
ing to the tissue size. An easy way to get rid of bubbles is to gently roll the tip of a pencil on the
coverslip.
52. Seal the coverslip with rubber cement (Figure 4).
Note: Make sure that the rubber cement straddles the edge of the coverslip.
CRITICAL: No proper coverslip sealing can produce probe evaporation, this could inter-fered in the hybridization reaction of probe to DNA.53. Place the slides on the ThermoBrite instrument and begin the denaturation-hybridization pro-
gram.a. Hybridize the slides incubating them from 12–16 h.Slide washes
Timing: 0.5 h and 8–16 h of incubation
Note: This step describes the protocol to perform the slide washes. Importantly the post-hy-
bridization washes can significantly affect the intensity and stability of the FISH signal. WhenSTAR Protocols 2, 100631, September 17, 2021 11
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OPEN ACCESS Protocolthe conditions are inappropriate, for instance by using inadequate temperature or time of
washing, background autofluorescence, unspecific signals or loss of the signals can appear.
Note: 30 min before you start the slide washes pre-warm the wash solution
54. Pre-warm the wash solution (0.43 SSC/0.3% NP-40) in a Coplin jar at 75C in a water bath for at
least 30 min prior to use.
55. Prepare a second container of 23 SSC/0.1% NP-40 at 20C–25C.
Note: The wash solutions are for single use application only.
56. Remove the rubber cement seal with forceps by gently pulling one of the corners of the seal
(Figure 4).
57. Remove the coverslip by soaking the slides in a Coplin jar with 23 SSC at 20C–25C.
Note: If the cover slip does not come out, it can be removed by tapping gently the slide in the
Coplin jar. If the coverslip is still stuck to the slide, slide the blade of a scalpel under one corner
of the slide and lift it gently before immersing the slide into the Coplin jar. This may need to be
repeated several times if the coverslip remains stuck.
58. Immediately place the slide into the 0.43 SSC/0.3% NP-40 wash solution at 73 G 1C.
a. Agitate the slide for 5 s.
b. Repeat with each slide.
Note: Do not wash more than four slides in each Coplin jar.
59. Wash the slide/s for 2 min into the 0.43 SSC/0.3% NP-40 wash solution at 73 G 1C.
60. Place the slide/s in the 23 SSC/0.1% NP-40 wash solution at 20C–25C.12a. Agitate the slide for 5 s and let them stand for 5 min.61. Air dry slide in the dark.
62. Apply to every slide 10 mL of counterstain dye (0.5 vol 40,6-diamidino-2-phenylindole DAPI II/0.5
vol antifade) and mount the slides with a 25350 mm coverslip.Selecting and enumerating cells for FISH analysis
Timing: 5 h
Note: This step describes how to select and enumerate cells within the selected target area,
for fish quantification and analysis.
63. Analyze the FISH signals at 10003 magnification using the appropriate filters.a. Select a representative area of the tissue with of good nuclear distribution (i.e., where indi-
vidual nuclei can be distinguished (Figure 5).
b. Using a 1003 objective and prescribed filters enumerate and record the number of signals
within each nucleus. Focus up and down to find all of the signal’s present in the nucleus.
c. Move to another representative area for enumeration until 200–300 tumor cells have been
enumerated.We performed FISH imaging on a Leica DM 5500B fluorescence microscope equipped with a 1003
oil-immersion objective, Leica DM DAPI, Aqua, Green and Orange fluorescence filter cubes and a
CCD camera (Photometrics SenSys camera) connected to a PC running the Zytovision image analysis
system (Applied Imaging Ltd., UK) with focus motor and Z stack software.STAR Protocols 2, 100631, September 17, 2021
Figure 5. Nuclei recognition by FISH: Selecting and enumerating cells within the selected target area
(A–C) Exemplary of three representative areas of tissue with a good nuclear distribution. Individual nuclei can be
distinguished and show a strong FISH signal together with weak or no fluorescence background. Only the tumor nuclei
with intact nuclear membrane, that do not overlap are used for the analysis. Images were captured using a Leica
DM5500B fluorescence microscope with a 1003 objective running the Cytovision v7.4 software. (A–C) White dashed
line highlights single nuclei that meet the described criteria; scale bars, 10 mm.
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OPEN ACCESSProtocolNote: The background should not contain particles that interfere with enumeration. In some
cases, fluorescent haze or glow may be noticeable outside of the nuclei, but it is acceptable as
long as the fluorescent haze/glow does not cover the nuclei and interfere with the
enumeration.
Note: Probe signal intensity: Analyze signal intensity on single color filters. The signals should
be bright, distinct, and easily evaluable. Signals should be compact, round or oval shapes.
Rubbish generally appears to be brighter and shinier compared to real signals, and back-
ground appear fuzzy and indistinct compared to real signal. Overly diffuse signals should
be avoided.
Note: FISH signal analysis can be donemanually or automatized by fluorescence image acqui-
sition scanning systems. For manual analysis two experienced scientists or technicians should
count the signals of each individual nucleus from 200–300 cells directly on the microscope us-
ing the appropriate filters. As an alternative, if the scientists or technicians have not enough
expertise in microscopy analysis, then representative images can be captured to complete
200–300 cells. In this case, FISH signals can be counted based on the captured images.CIN analysis
Timing: 0.5–1 h
Note: average time for an experienced observer to visualize one FISH probe slide.
The CIN score (CS) is a metric devised to quantify the alterations (copy number changes) detected in
a sample, that would lead to chromosomal heterogeneity and could drive different cancer patterns
from initiation, progression, evolution and drug resistance patterns. Depending on the size of the
genomic alteration it can be classified as: structural CIN if it involves translocations, inversions, de-
letions and amplifications that range from genes to complete chromosome arms (Geigl et al., 2008);
or as numerical CIN if it involves whole chromosomes gain or losses.
In order to quantify these alterations, there are many available techniques, with their own pros and
cons (Lepage et al., 2019). Based in our research paper (Brandt et al., 2018), we have detailed here
how to measure the CS by FISH.
To perform manual FISH analysis, visualize, count and annotate every FISH signal of each nucleus
under the microscope using its specific filter. Computes a summary index of the FISH signals basedSTAR Protocols 2, 100631, September 17, 2021 13
Figure 6. Pepsin treatment calibration
Representative pictures were serial sections of FFPE tissues were incubated at different time points with ready to use
pepsin to test the correct time for digestion. Incubations were performed at (A) 30 min, (B) 45 min, (C) 60 min and (D)
120 min (only Dapi channel are shown) before probe hybridization. The total time of wash buffer incubation and
hybridization was equal for all conditions. Images were captured using a Leica DM5500B fluorescence microscope
with a 1003 objective running the Cytovision v7.4 software. (A–D) scale bars, 10 mm.
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OPEN ACCESS Protocolon all examined cells. Alternative FISH analysis can be done counting signals on captured images.
Different capture platforms are available; some examples are Cytovision (Leica) or Metafer (Meta-
Systems). This step describes the protocol to perform the FISH analysis on captured images.
64. Take representative images from several tissue areas that account for at least 200–300 nuclei
signals. Select only intact nuclei that are not folded, overlapped or obstructed by debris (as
described in the FISH protocol section) (Figure 6).
65. Evaluate and annotate the total number of signals of each nucleus for any of the probes de-
signed before to start with the following one to determine the final 200–300 nuclei count.
Note: CS is used to describe the aneuploidies within a given sample. When calculated for a
single chromosome is known as CS, and for several stained chromosomes as CSC (CIN score
combined).
66. Calculate the CS value for each chromosome probe [e.g., CS for chromosomes 1, 2, or 3 (CS1,
CS2, or CS3, respectively)] of a given sample, applying the following formula:
CSChrp = 1
.
n
X
eChrpoChrp
Where:
n : accounts for the number of nuclei analyzed.
oChrp: are the observed signals for a specific chromosome (p).14 STAR Protocols 2, 100631, September 17, 2021
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OPEN ACCESSProtocoleChrp: are the value that corresponds to the expected signals for the given chromosome (p).
The ep value is usually equal to 2, that is the number of signals for a wild type cell (2n).
67. Sum each of the individual CS values to obtain the corresponding CSC for a given sample, as
follows:
CSC =

CSChrp + CSChrq + CSChrr
Note: Alternatively, an overall mean CS can be calculated for each sample by summing the
CSC for each nucleus analyzed withing a sample and dividing by the total number of nuclei
evaluated:
CSc = 1
.
n
X
epop + eqoqNote: As a reference, CSc = 0 indicates a wild type nucleus has been analyzed. By the con-
trary, a value higher than 0 ðCSc > 0Þ indicates gains/losses of chromosomes and hence, a
specific degree of chromosomal instability. The higher the value, the greater the instability.EXPECTED OUTCOMES
As described, the CS is a metric devised to quantify chromosomal alterations in the samples through
the use of two probes hybridized on each nucleus analyzed. We assessed CS aneuploidies of chro-
mosomes 11 and 2, and described that CINmay be a driver for inflammation-induced tumorigenesis
(Brandt et al., 2018). Figures 7 illustrates FISH probe hybridization of chromosomes to visualize
frequently occuring chromosomal abnormalities and its corresponding FISH signaling pattern.
Figure 8 illustrates probe hybridization of chromosomes 11 and 2 used in (Brandt et al., 2018),
and frequent scenarios during CIN quantification.LIMITATIONS
CIN refers to an ongoing rate of change that drives cell-to-cell heterogeneity rather than a static
state such as aneuploidy. For that reason, it is important to assess CIN at the single cell level using
techniques that are capable of capturing the full spectrum of heterogeneity and CIN existing within a
given sample. In this sense, FISH allows the quantitative assessment of cell-to-cell heterogeneity
within a given population analyzing karyotypic heterogeneity. Although, FISH analysis enables the
labeling of specific genes, regions, or whole chromosomes, is amenable to automation and multi-
plexing, and can be applied to fixed samples. Its main limitation is that it only detects events
involving the labeled locus/chromosomes; multiplexing depends on the availability of spectrally
distinct fluorophores. Metaphase G-banding analysis may be helpful in characterizing abnormal
chromosomal patterns, however only a low number of cells can be analyzed using this approach.
As an alternative, single-cell high throughput sequencing could also be useful, but this approach
considerably increases the cost of analysis.TROUBLESHOOTING
Problem 1
Low DNA yield
Step: BAC DNA isolationPotential solutions
Poor aeration of culture: The optimal culture volume to air volume ratio is 1:4. For best aeration
vented or gas-permeable seal culture flasks can be used. (steps 1–5)STAR Protocols 2, 100631, September 17, 2021 15
Figure 7. Schematic representation of chromosomal abnormalities visualized after FISH assay
(A) Schematic representation at genomic level of a control diploid cell containing a pair of chromosomes, and its
corresponding normal FISH signal pattern is illustrated in the second column (Top).
(B) (Top) Schematic representation of the amplification of a specific targeted locus. Gene amplification can be
observed both at genomic level and through FISH signaling pattern. (Bottom) Schematic representation of locus
deletion, upon chromosome breaks. Gene deletion can be observed both at genomic level and through FISH
signaling pattern.
(C) Polyploidy where the probe signal shows multiple sets of chromosomes, observed by three pair of fused genes.
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OPEN ACCESS ProtocolIncomplete neutralization: Cell debris will float to the surface. Ensure that neutralization is complete
prior to centrifugation. Invert the tube an additional 2–3 times. (step 7)
Incomplete elution: Pre-warming water to 50C prior to elution can increase the DNA yield. (step 16)Problem 2
Low DNA quality
Step: FISH preparation: BAC DNA isolationPotential solutions
Incomplete neutralization generates poor quality of supernatant, and results in cell debris contam-
ination during the following steps of the protocol. Make sure the neutralization is complete prior to
centrifugation. Additionally, invert the tube 2–3 times. (step 7)16 STAR Protocols 2, 100631, September 17, 2021
Figure 8. Probe hybridization and FISH signaling pattern
(A) Schematic representation at genomic level of probe hybridization for the detection of chromosomal aberrations in
chromosomes 2 and 11.
(B) Schematic representation of fluorescent probe visualization in interphase nucleus after FISH assay, where the
green signal corresponds to chromosome 2 and the red signal corresponds to 11. From top to bottom, the first nuclei
represent a control diploid cell with two copies of each chromosome (two red signals, and two green signals). The
second nuclei represent an abnormal signaling pattern where chromosome aneuploidy can be visualized (two green
signals and four red signals).
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OPEN ACCESS
STAR Protocols 2, 100631, September 17, 2021 17
Protocol
Figure 8. Continued
(C) Schematic representation at genomic level of probes hybridization for the detection of chromosomal aberrations,
in chromosomes 2, 9 and 19.
(D) Schematic representation of fluorescent probe visualization in interphase nucleus after FISH assay, where the
yellow signal corresponds to chromosome 2, the red signal corresponds to chromosome 9, and the green signal
corresponds to chromosome 19. From top to bottom, the first nuclei represent a control diploid cell with two copies of
each chromosome (two yellow signals, two red signals, and two green signals). The second nuclei represent an
abnormal signaling pattern where chromosome aneuploidy can be visualized (four yellow signals, three red signals
two green signals and).
(E) Schematic representation at genomic level of probes hybridization for the detection of chromosomal aberrations,
in chromosomes 17 and 11.
(F) Schematic representation of fluorescent probe visualization in interphase nucleus after FISH assay, where the red
signal corresponds to a specific locus of chromosome 17 and the green signal corresponds to a specific locus of
chromosome 11. From top to bottom, the first nuclei represent a control diploid cell with two copies of each
chromosome locus (two red signals, and two green signals). The second nuclei represent an abnormal signaling
pattern where the deletion of the analyzed locus can be visualized (two green signals and four red signals). (B, D, and
F) Calculations for CSc score are represented in blue squares (normal signal pattern) and red squares (abnormal signal
pattern), for each represented example.
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OPEN ACCESS ProtocolRNA contamination: Ensure that RNase A has enough time to degrade RNA by allowing lysate to
incubate at 20C–25C an additional 3–5 min (step 13)
Genomic DNA contamination: Improper handling. Genomic DNA contamination is usually caused
by excessive mechanical shearing during the lysis and neutralization steps. Prolonged lysis or incom-
plete mixing of neutralization buffer may affect genomic DNA contamination. (steps 6 and 7)Problem 3
Low FISH probe yield
Step: FISH preparation: BAC DNA isolation (steps 20–22 )Potential solutions
Ensure that the length of the probe is adequate. Short fragment sizes, smaller than 200–500 bp gives
low yield. Check if enough template (minimum 1 mg) was used for labeling.
DNAse I might be inhibited by contaminants such as amines. Care should be taken to ensure that all
impurities which might interfere in the labeling reaction have been removed from the DNA. Phenol-
chloroform DNA purification can enhance DNA purity.Problem 4
FISH: Weak or no FISH signal
Step: Stated at each potential solutionPotential solutions
Ensure you obtained an adequate FISH probe yield, efficient dye incorporation and the expected
probe length. You might need to optimize quality of the template, amount of the starting material,
reaction temperatures and time. (step 17)
Ensure that the pre-treatment steps were made correctly; that the temperature of the pre-treatment so-
lutions is optimal; andmake sure to replace the pre-treatment solution after every batch (Figure 5). (steps
35–39)
Ensure the correct time for pepsin digestion. Importantly, the user may need to optimize their own
times depending on the extent of fixation, age of archived tissue or the type of tissue to be digested.18 STAR Protocols 2, 100631, September 17, 2021
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OPEN ACCESSProtocolUse a start digestion time of 15 min, however increasing to 30 or 45 min incubation times can
improve signal intensity or autofluorescence background in some samples (Figure 6). (step 42)
Ensure the temperature of denaturation. The denaturation temperature is set at 75C; however,
increasing it to 80C can improve the signal strength. Time of denaturation can also be adjusted.
(step 50)
Use 10 to 15 ml of probe solution and increase the volume when using larger sections. Ensure a good
seal with rubber glue to prevent evaporation. Ensure that no air bubbles were trapped under the
coverslip before hybridization. (steps 52 and 53)
Temperature and pH of post-hybridization washes also influence in the intensity of the FISH signals;
increasing the temperature increases the stringency, and the pH determines the availability of the
positive ions that counteract the repulsive negative force between the DNA backbones of both
the probe and target. Ensure that the wash solutions was prepared correctly and had the proper
pH and temperature. (step 55)
Prolonged storage of FISH probes can lead to substantial probe degradation. Probesmust be stored
at 20C for no longer than a year.Problem 5
FISH: High slide background
Step: Pretreatment of formalin-fixed paraffin-embedded (FFPE) tissue sectionPotential solution
Ensure that the wash solutions was prepared correctly, having the proper pH and temperature. Re-
move the coverslip before immersing the slides in the wash solution. (steps 55–58)
High backgroundmay be due to insufficient removal of material during the pre-treatment steps. The
extent of fixation, the age of archived tissue or the type of tissue can affect the results of FISH hybrid-
ization. Adjustment of digestion time can improve autofluorescence background in some samples
(Figure 6). (Step 42)
High background may be due incorrect sealing off the slides with rubber cement during the pre-
treatment and co-denaturation steps, since this allows the solution to evaporate and the tissue to
dry out. Placing the slides in the incubator to allow the rubber cement to dry before these steps
can reduce this effect. (steps 52 and 53)Problem 6
FISH: Degraded tissue
Step: Pretreatment of formalin-fixed paraffin-embedded (FFPE) tissue sectionPotential solutions
Pre-treatment steps can render the tissue softer, and it may easily fall off or get degraded. The size of
the tissue gives a good indication as to the fragility of the tissue, so this should be taken into account
to decide the incubation time of the pre-treatment solution. Small tissues should be incubated for
10 min (step 38–40)
Reducing pepsin enzyme treatment on the sample may also avoid this problem. (step 42)STAR Protocols 2, 100631, September 17, 2021 19
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OPEN ACCESS ProtocolRESOURCE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to and will be ful-
filled by the lead contact, Dr. Nabil Djouder (Correspondence: ndjouder@cnio.es).
Further technical information should be directed to and will be fulfilled by the technical contact, Dra.
Sandra Rodriguez-Perales (Correspondence: srodriguezp@cnio.es).
Materials availability
Plasmids generated in this study and materials are available upon request from the lead or technical
contact; the sharing of materials described in this work could be subject to standard Material Trans-
fer Agreements.
Data and code availability
Datasets and code generated or analyzed in this study can be found in the key resources table.
ACKNOWLEDGMENTS
This work was funded by grants to N.D. from the State Research Agency (AEI, 10.13039/
501100011033) from the Spanish Ministry of Science and Innovation (projects SAF2016-76598-R,
SAF2017-92733-EXP, RTI2018-094834-B-I00, and RED2018-102723-T), cofounded by European
Regional Development Fund (ERDF). S.R-P. is supported by grants from the Spanish National
Research and Development Plan, Instituto de Salud Carlos III, and Spanish Ministry of Science
and Innovation cofounded by ERDF (PI17/02303; DTS19/00111 and BIO2017-91272-EXP) and by
the Asociacio´n Espan˜ola Contra el Ca´ncer (AECC). R.T.-R. was supported by a postdoctoral fellow-
ship from the AECC. This work was developed at the CNIO funded by the Health Institute Carlos III
(ISCIII) and the Spanish Ministry of Science and Innovation.
AUTHOR CONTRIBUTIONS
R.T.-R. and T.P.G. wrote the protocol with M.B. and M.M.-L.; T.P.G. made the figures and graphical
abstract; R.T-R, T.P.G, S.R.-P. andN.D reviewed, edited and corrected the protocol; S.R.-P. andN.D.
supervised the study, and secured funding. N.D. designed the study and was in charge of the project
administration.
DECLARATION OF INTERESTS
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