3. For smFISH performed using Quasar 670 (CY5 filter) or
Quasar 570 (CY3 filter), use an exposure time between
750 and 1000 ms to acquire each Z plane and 100% light
power (120 W Mercury Arc lamp).
4. For IF performed using Alexa 647 (CY5 filter), Alexa
555 (CY3 filter), or Alexa 488 (FITC filter) dyes, expose each
Z plane 300–500 ms, and 100% light power.
5. For the DAPI channel use 25–50 ms exposure and 12.5% light
source power.
6. Acquire the differential interference contrast (DIC) image for
50–100 ms on a single plane (see Note 2).
Tub1 protein
(Alexa 647)
CLN2 mRNA
(Quasar 570)
a
b
CLN2 mRNA expression during the cell cycle
Tub1 protein
CLN2 mRNA
DIC
DAPI
MERGE
G2
G1(early)
G1(early)
G1(late)
G1(late)
G1
S
G2
M
bud
mother
CLN2 mRNA smFISH
Tub1 IF
DIC
DAPI
c
MAX projection
of Z-stacks (~20)
covering the cells
thickness
Fig. 3 smFISH-IF for the cyclin CLN2 mRNA and the tubulin protein. (a) Schematic representation of CLN2
mRNA expression during the cell cycle. Yellow dots represent CLN2 mRNA in the cytoplasm and transcription
sites in the nucleus, which are brighter than the cytoplasmic mRNAs. Tubulin co-localizes with the spindle
pole body, which is duplicated during S phase. The bud emergence starts during S phase and ends with the
formation of the daughter cell. During anaphase, the microtubules stretch between the mother and the
daughter cell. The CLN2 mRNA is transcribed during late G1 and it diffusely localizes in the cytoplasm. (b)
MERGE maximally projected image: CLN2 mRNA smFISH Quasar 570 (yellow), tubulin IF Alexa 647 (magenta),
and DAPI (blue) merged to a single-plane DIC image (gray). The cell cycle phase of few representative cells is
indicated in the image. Scale bar 10 μm. (c) Maximally projected channels from image shown in (b) are
individually displayed
Detection of mRNA and Proteins in Single Cells by smFISH-IF
61
Quasar 570 (CY3 filter), use an exposure time between
750 and 1000 ms to acquire each Z plane and 100% light
power (120 W Mercury Arc lamp).
4. For IF performed using Alexa 647 (CY5 filter), Alexa
555 (CY3 filter), or Alexa 488 (FITC filter) dyes, expose each
Z plane 300–500 ms, and 100% light power.
5. For the DAPI channel use 25–50 ms exposure and 12.5% light
source power.
6. Acquire the differential interference contrast (DIC) image for
50–100 ms on a single plane (see Note 2).
Tub1 protein
(Alexa 647)
CLN2 mRNA
(Quasar 570)
a
b
CLN2 mRNA expression during the cell cycle
Tub1 protein
CLN2 mRNA
DIC
DAPI
MERGE
G2
G1(early)
G1(early)
G1(late)
G1(late)
G1
S
G2
M
bud
mother
CLN2 mRNA smFISH
Tub1 IF
DIC
DAPI
c
MAX projection
of Z-stacks (~20)
covering the cells
thickness
Fig. 3 smFISH-IF for the cyclin CLN2 mRNA and the tubulin protein. (a) Schematic representation of CLN2
mRNA expression during the cell cycle. Yellow dots represent CLN2 mRNA in the cytoplasm and transcription
sites in the nucleus, which are brighter than the cytoplasmic mRNAs. Tubulin co-localizes with the spindle
pole body, which is duplicated during S phase. The bud emergence starts during S phase and ends with the
formation of the daughter cell. During anaphase, the microtubules stretch between the mother and the
daughter cell. The CLN2 mRNA is transcribed during late G1 and it diffusely localizes in the cytoplasm. (b)
MERGE maximally projected image: CLN2 mRNA smFISH Quasar 570 (yellow), tubulin IF Alexa 647 (magenta),
and DAPI (blue) merged to a single-plane DIC image (gray). The cell cycle phase of few representative cells is
indicated in the image. Scale bar 10 μm. (c) Maximally projected channels from image shown in (b) are
individually displayed
Detection of mRNA and Proteins in Single Cells by smFISH-IF
61