6. Excessive fixation will reduce the efficiency of yeast cell wall
removal during the Lyticase treatment.
7. Duration on Lyticase treatment varies with yeast strain and
treatment. Over-digestion will damage cell morphology and
will preclude accurate counting of single molecules.
8. 70% Ethanol allows to store the cells for several months. It also
perforates the cell membrane helping the penetration of the
smFISH probes during hybridization. Although a 20-min
incubation in 70% ethanol at À20
C should be enough for
cell membrane permeabilization, we usually do an overnight
incubation.
9. At this stage a phase-contrast microscope should be used to
check if the cells are well attached to the coverslip. If cells
detach at this step from the coverslips, the coverslip may not
have been completely dry after poly-L-lysine treatment.
10. The pre-hybridization and the hybridization solutions contain
10% (vol/vol) formamide in 2Â SSC. This formamide concentration is optimal for ~20-nt-long probes. If using longer
probes of 50 nt, the recommended formamide concentration
is 50%.
11. To perform smFISH for two mRNAs simultaneously, add for
each coverslip an equal amount (0.125 μL of a 25 μM stock) of
the two probe mixes in 5 μL of the DNA/RNA competitor
and lyophilize. Continue the protocol from paragraph Subheading 3.3, step 4. If only one smFISH probe set is used we
recommend the use of probes labeled with Quasar 570/Cy3
fluorophore. If a two-color smFISH is performed, we recommend the use of probes labeled with Quasar 670/Cy5 and
Quasar 570/Cy3 dyes (Table 1).
12. Do not extend the washing time; otherwise the smFISH signal
will reduce significantly.
13. As anti-alpha-tubulin antibody we use a mouse monoclonal
antibody from Thermo Fisher (#236-10501). The use of polyclonal antibodies from other brands led to higher background
in the IF channel under our conditions. Resuspend the lyophilized stock in 2 mM Na azide, 1% BSA, and PBS. The stock
concentration is 160 ng/mL. Dilute the antibody 1:1000 in IF
solution.
14. The secondary antibody stock concentration is 2 mg/mL.
Dilute the antibody 1:1500 in IF solution.
15. Let nail polish dry before imaging by microscopy in order to
avoid damaging the objective.
16. Slides can be stored at 4
C for a few days or at À20
C for
months in the dark.
66
Evelina Tutucci and Robert H. Singer
removal during the Lyticase treatment.
7. Duration on Lyticase treatment varies with yeast strain and
treatment. Over-digestion will damage cell morphology and
will preclude accurate counting of single molecules.
8. 70% Ethanol allows to store the cells for several months. It also
perforates the cell membrane helping the penetration of the
smFISH probes during hybridization. Although a 20-min
incubation in 70% ethanol at À20
C should be enough for
cell membrane permeabilization, we usually do an overnight
incubation.
9. At this stage a phase-contrast microscope should be used to
check if the cells are well attached to the coverslip. If cells
detach at this step from the coverslips, the coverslip may not
have been completely dry after poly-L-lysine treatment.
10. The pre-hybridization and the hybridization solutions contain
10% (vol/vol) formamide in 2Â SSC. This formamide concentration is optimal for ~20-nt-long probes. If using longer
probes of 50 nt, the recommended formamide concentration
is 50%.
11. To perform smFISH for two mRNAs simultaneously, add for
each coverslip an equal amount (0.125 μL of a 25 μM stock) of
the two probe mixes in 5 μL of the DNA/RNA competitor
and lyophilize. Continue the protocol from paragraph Subheading 3.3, step 4. If only one smFISH probe set is used we
recommend the use of probes labeled with Quasar 570/Cy3
fluorophore. If a two-color smFISH is performed, we recommend the use of probes labeled with Quasar 670/Cy5 and
Quasar 570/Cy3 dyes (Table 1).
12. Do not extend the washing time; otherwise the smFISH signal
will reduce significantly.
13. As anti-alpha-tubulin antibody we use a mouse monoclonal
antibody from Thermo Fisher (#236-10501). The use of polyclonal antibodies from other brands led to higher background
in the IF channel under our conditions. Resuspend the lyophilized stock in 2 mM Na azide, 1% BSA, and PBS. The stock
concentration is 160 ng/mL. Dilute the antibody 1:1000 in IF
solution.
14. The secondary antibody stock concentration is 2 mg/mL.
Dilute the antibody 1:1500 in IF solution.
15. Let nail polish dry before imaging by microscopy in order to
avoid damaging the objective.
16. Slides can be stored at 4
C for a few days or at À20
C for
months in the dark.
66
Evelina Tutucci and Robert H. Singer