Day 1:
1. Pipette out the cell culture medium from the 8-well chambered
cover glass and incubate each well with 350 μL PBS prewarmed
to 37
C for 5 min.
2. Pipette out the PBS and add 250 μL of 4% PFA pre-warmed to
37
C per well. Incubate for 30 min at RT to fix the cells.
3. Pipette out the PFA and incubate each well of the cells in
350 μL PBS at RT for 5 min.
4. Pipette out the PBS and permeabilize the cells in 250 μL of
0.5% NP-40 at RT for 10 min.
5. Pipette out the NP-40 and incubate the cells in 350 μL PBS at
RT for 5 min.
6. Pipette out the PBS and incubate each well of the samples with
250 μL of TAMRA-labeled FISH probes (diluted in DNA
hybridization buffer to a final concentration of 100 nM).
7. Wrap the chambered cover glass with sealing film to minimize
evaporation. Protect from light and incubate at 37
C overnight (>16 h).
Day 2:
1. Gently pipette out unbound DNA FISH probes.
2. Wash cells in wash buffer followed by 2Â SSC, 1Â SSC, and
0.2Â SSC to remove unhybridized probes. Do not let the
samples dry between washes.
3. Incubate cells in PBS containing 100 ng/mL DAPI to stain the
nucleus.
4. Use DAPI, TRITC, and Cy5 filter sets to image the nuclear,
FISH, and MB signals, respectively. For FISH and MB signals,
3D image stacks should be obtained at an increment of
0.25 μm in the z-direction. Representative maximum intensity
projection images of the FISH and MB signals are shown in
Fig. 2a (see Note 22).
3.4 Validation
of CRISPR/MB Signals
by Immunofluorescence (IF)
In some cases, IF is also an effective method to validate the labeling
accuracy of CRISPR/MB, as the locus of interest, such as the major
satellite repeats within centromere, is bound by many endogenous
DNA-binding proteins. Thus, when antibodies that can specifically
bind to the cognate binding proteins of the locus are used, the
resulting IF signal can be used to validate the accuracy of the
CRISPR/MB signal. The detailed protocol for IF is shown below.
1. Gently pipette out the cell culture medium from the 8-well
chambered cover glass and incubate each well with 350 μL PBS
pre-warmed to 37
C for 5 min.
Visualizing Genomic Loci by CRISPR/MB
365
1. Pipette out the cell culture medium from the 8-well chambered
cover glass and incubate each well with 350 μL PBS prewarmed
to 37
C for 5 min.
2. Pipette out the PBS and add 250 μL of 4% PFA pre-warmed to
37
C per well. Incubate for 30 min at RT to fix the cells.
3. Pipette out the PFA and incubate each well of the cells in
350 μL PBS at RT for 5 min.
4. Pipette out the PBS and permeabilize the cells in 250 μL of
0.5% NP-40 at RT for 10 min.
5. Pipette out the NP-40 and incubate the cells in 350 μL PBS at
RT for 5 min.
6. Pipette out the PBS and incubate each well of the samples with
250 μL of TAMRA-labeled FISH probes (diluted in DNA
hybridization buffer to a final concentration of 100 nM).
7. Wrap the chambered cover glass with sealing film to minimize
evaporation. Protect from light and incubate at 37
C overnight (>16 h).
Day 2:
1. Gently pipette out unbound DNA FISH probes.
2. Wash cells in wash buffer followed by 2Â SSC, 1Â SSC, and
0.2Â SSC to remove unhybridized probes. Do not let the
samples dry between washes.
3. Incubate cells in PBS containing 100 ng/mL DAPI to stain the
nucleus.
4. Use DAPI, TRITC, and Cy5 filter sets to image the nuclear,
FISH, and MB signals, respectively. For FISH and MB signals,
3D image stacks should be obtained at an increment of
0.25 μm in the z-direction. Representative maximum intensity
projection images of the FISH and MB signals are shown in
Fig. 2a (see Note 22).
3.4 Validation
of CRISPR/MB Signals
by Immunofluorescence (IF)
In some cases, IF is also an effective method to validate the labeling
accuracy of CRISPR/MB, as the locus of interest, such as the major
satellite repeats within centromere, is bound by many endogenous
DNA-binding proteins. Thus, when antibodies that can specifically
bind to the cognate binding proteins of the locus are used, the
resulting IF signal can be used to validate the accuracy of the
CRISPR/MB signal. The detailed protocol for IF is shown below.
1. Gently pipette out the cell culture medium from the 8-well
chambered cover glass and incubate each well with 350 μL PBS
pre-warmed to 37
C for 5 min.
Visualizing Genomic Loci by CRISPR/MB
365