tools. If the smFISH worked well, the distribution of the
intensities of the cytoplasmic spots should resemble a Gaussian
distribution (Fig. 6c). Nuclear spots are usually brighter than
cytoplasmic spots and they can be automatically filtered based
on their intensity. The smFISH quantifications can be correlated with the IF, for instance, by creating outlines for specific
stages of the cell cycle, based on the alpha-tubulin expression
and pattern (Note 18).
4 Notes
1. FISH probes were custom designed against ASH1 and CLN2
by using the Stellaris RNA FISH Probe Designer (Biosearch
Technologies) freely available online at http://www.
biosearchtech.com/stellarisdesigner. Each smFISH probe mix
is shipped as a dried set containing up to 48 individual smFISH
probes, mixed in an equimolar ratio. We eliminate probes that
have more than 85% homology with other mRNAs to avoid
high background signal in the smFISH channel.
2. For smFISH imaging we use an Olympus BX-63 epifluorescence microscope equipped with Ultrasonic stage and UPlanApo 100Â, 1.35NA oil-immersion objective (Olympus). An
X-Cite 120 PC Lamp (EXFO), an ORCA-R2 Digital Interline
CCD Camera (C10600-10B; Hamamatsu; 6.45 μm-pixel size)
mounted using U-CMT and 1X-TVAD Olympus c-Mount
Adapters, and zero-pixel shift filter sets: DAPI-5060C-Zero,
FITC-5050-000, Cy3-4040C-Zero, and Cy5-4040C-Zero
from Semrock. We acquired data using 41 optical sections
with a z-step size of 0.2 μm. MetaMorph (Molecular Devices)
software was used for instrument control as well as image
acquisition.
3. For smFISH analysis we recommend using FISH-quant [31], a
free software developed in the MATLAB programming language (MathWorks). Download the FISH-quant package
(http://code.google.com/p/fish-quant/) together with the
MCRInstaller, which allows one to run a MATLAB algorithm
without separately installing MATLAB onto the computer.
4. For imaging processing we recommend the free software Fiji
(Java software for image processing analysis; freely available at
https://fiji.sc/) [32].
5. Keep the cells growing in exponential phase (OD 600 <1) at all
times. At this density the autofluorescence of the cells is
minimal.
Detection of mRNA and Proteins in Single Cells by smFISH-IF
65
intensities of the cytoplasmic spots should resemble a Gaussian
distribution (Fig. 6c). Nuclear spots are usually brighter than
cytoplasmic spots and they can be automatically filtered based
on their intensity. The smFISH quantifications can be correlated with the IF, for instance, by creating outlines for specific
stages of the cell cycle, based on the alpha-tubulin expression
and pattern (Note 18).
4 Notes
1. FISH probes were custom designed against ASH1 and CLN2
by using the Stellaris RNA FISH Probe Designer (Biosearch
Technologies) freely available online at http://www.
biosearchtech.com/stellarisdesigner. Each smFISH probe mix
is shipped as a dried set containing up to 48 individual smFISH
probes, mixed in an equimolar ratio. We eliminate probes that
have more than 85% homology with other mRNAs to avoid
high background signal in the smFISH channel.
2. For smFISH imaging we use an Olympus BX-63 epifluorescence microscope equipped with Ultrasonic stage and UPlanApo 100Â, 1.35NA oil-immersion objective (Olympus). An
X-Cite 120 PC Lamp (EXFO), an ORCA-R2 Digital Interline
CCD Camera (C10600-10B; Hamamatsu; 6.45 μm-pixel size)
mounted using U-CMT and 1X-TVAD Olympus c-Mount
Adapters, and zero-pixel shift filter sets: DAPI-5060C-Zero,
FITC-5050-000, Cy3-4040C-Zero, and Cy5-4040C-Zero
from Semrock. We acquired data using 41 optical sections
with a z-step size of 0.2 μm. MetaMorph (Molecular Devices)
software was used for instrument control as well as image
acquisition.
3. For smFISH analysis we recommend using FISH-quant [31], a
free software developed in the MATLAB programming language (MathWorks). Download the FISH-quant package
(http://code.google.com/p/fish-quant/) together with the
MCRInstaller, which allows one to run a MATLAB algorithm
without separately installing MATLAB onto the computer.
4. For imaging processing we recommend the free software Fiji
(Java software for image processing analysis; freely available at
https://fiji.sc/) [32].
5. Keep the cells growing in exponential phase (OD 600 <1) at all
times. At this density the autofluorescence of the cells is
minimal.
Detection of mRNA and Proteins in Single Cells by smFISH-IF
65