S. cerevisiae [27]. To distinguish the different phases of the cell
cycle we performed IF against the microtubule component alphatubulin, encoded by the gene TUB1. Because during mitosis the
microtubules extend from the spindle pole bodies between the
mother and daughter cell [28], monitoring tubulin expression
can provide precise information about the cell cycle phase that can
be correlated with mRNA expression.
Few smFISH-IF protocols have been recently published for
mammalian or insect cells [29, 30]. Here, we present our current
smFISH-IF protocol for the model organism S. cerevisiae, which
can be used to visualize up to three colors at the time (i.e., two
mRNAs and one protein). The protocol is performed over 2 days,
followed by imaging and analysis. The major steps are DAY
1, (1) coverslip coating (1 h); (2) growth, fixation, and permeabilization of the cells (overnight growth + ~4-h processing time);
DAY 2, (3) smFISH hybridization and washes (4 h); (4) immunofluorescence and mounting (3 h); DAY 3, (5) image acquisition;
and (6) imaging analysis (time depends on the aim of the
experiment).
This approach can be applied to many different biological
questions and model organisms; the limitation is the restricted
number and the specificity of primary antibodies available, which
can be circumvented by using tagged proteins. Subheading 4
describes alternative approaches to simultaneously visualize more
than one mRNA and protein. These considerations may be useful as
a primer to develop or troubleshoot smFISH-IF protocols for
other yeast species or other model organisms.
2 Materials
Prepare all solutions using double-distilled ultrapure RNase-free
water (DDW). Paraformaldehyde and deionized formamide are
hazardous solutions. Wear protective gloves and handle them
under a fume hood.
1. 0.1 N HCl.
2. 70% Ethanol.
3. Noncoated coverslips: 0.13–0.17 mm thick; diameter 18 mm.
4. Twelve-well culture dish.
5. 0.01% Poly-L-lysine.
6. 15 and 50 mL plastic tubes.
7. 1.5 mL Tubes.
8. Refrigerated centrifuges for 1.5 mL or 50 mL tubes.
9. Parafilm.
Detection of mRNA and Proteins in Single Cells by smFISH-IF
53
cycle we performed IF against the microtubule component alphatubulin, encoded by the gene TUB1. Because during mitosis the
microtubules extend from the spindle pole bodies between the
mother and daughter cell [28], monitoring tubulin expression
can provide precise information about the cell cycle phase that can
be correlated with mRNA expression.
Few smFISH-IF protocols have been recently published for
mammalian or insect cells [29, 30]. Here, we present our current
smFISH-IF protocol for the model organism S. cerevisiae, which
can be used to visualize up to three colors at the time (i.e., two
mRNAs and one protein). The protocol is performed over 2 days,
followed by imaging and analysis. The major steps are DAY
1, (1) coverslip coating (1 h); (2) growth, fixation, and permeabilization of the cells (overnight growth + ~4-h processing time);
DAY 2, (3) smFISH hybridization and washes (4 h); (4) immunofluorescence and mounting (3 h); DAY 3, (5) image acquisition;
and (6) imaging analysis (time depends on the aim of the
experiment).
This approach can be applied to many different biological
questions and model organisms; the limitation is the restricted
number and the specificity of primary antibodies available, which
can be circumvented by using tagged proteins. Subheading 4
describes alternative approaches to simultaneously visualize more
than one mRNA and protein. These considerations may be useful as
a primer to develop or troubleshoot smFISH-IF protocols for
other yeast species or other model organisms.
2 Materials
Prepare all solutions using double-distilled ultrapure RNase-free
water (DDW). Paraformaldehyde and deionized formamide are
hazardous solutions. Wear protective gloves and handle them
under a fume hood.
1. 0.1 N HCl.
2. 70% Ethanol.
3. Noncoated coverslips: 0.13–0.17 mm thick; diameter 18 mm.
4. Twelve-well culture dish.
5. 0.01% Poly-L-lysine.
6. 15 and 50 mL plastic tubes.
7. 1.5 mL Tubes.
8. Refrigerated centrifuges for 1.5 mL or 50 mL tubes.
9. Parafilm.
Detection of mRNA and Proteins in Single Cells by smFISH-IF
53