association [16]. Before the smFISH-stained coverslips are
processed for immunofluorescence, we recommend that they
be extensively washed with PBS to fully remove any formamide, which can reduce the efficiency of immunolabeling. For
the immunostaining, it is best to use purified antibodies and to
avoid whole-serum preparations as these tend to contain
RNase which will reduce the smFISH signal. Many protocols
use whole serum to reduce background staining; however we
have found that this can be done by including 1% bovine serum
albumin and 0.1% Triton-X100 to the primary and secondary
antibody solutions.
24. We use Nikon Imaging Software (NIS-Elements) to collect and
process the micrographs.
25. To ensure that the fluorescence intensities between fields are
comparable, the exposure time should remain constant
between different fields of view and different coverslips. Furthermore, all of the imaging of one experimental set should be
done in one sitting to minimize any day-to-day fluctuations in
the intensity of the excitation light source.
26. To identify spots, use the Spot Detection Module on
NIS-Elements (Fig. 3) using the Bright, Clustered Method
option. For settings, for the “Typical Diameter” we use
0.32 μm, and for the “Remove Dim Objects” we set the level
so that few to no spots are selected in images taken in parallel of
cells where we either omit the smFISH probes or stain with
scrambled probes (see Note 19). This is essential as there is
always a low level of staining caused by autofluorescence or
probes binding to suboptimal targets. The total number of
spots in whole cell and nuclear ROIs are counted automatically
using the “Automated Measurement Results” and the number
of spots in the cytoplasm is calculated by subtracting the
nuclear spots from the total cell spots. The number of nuclear
foci can be used as a control to ensure that the unextracted and
digitonin-extracted cells have been stained equally well. Nuclei
should be impervious to digitonin extraction [20].
References
1. Cui XA, Palazzo AF (2014) Localization of
mRNAs to the endoplasmic reticulum. Wiley
Interdiscip Rev RNA 5:481–492
2. Rapoport TA (2007) Protein translocation
across the eukaryotic endoplasmic reticulum
and bacterial plasma membranes. Nature
450:663–669
3. Walter P, Ibrahimi I, Blobel G (1981) Translocation of proteins across the endoplasmic
reticulum. I. Signal recognition protein (SRP)
binds to in-vitro-assembled polysomes synthesizing secretory protein. J Cell Biol
91:545–550
4. Gilmore R, Blobel G, Walter P (1982) Protein
translocation
across
the
endoplasmic
reticulum. I. Detection in the microsomal
membrane of a receptor for the signal recognition particle. J Cell Biol 95:463–469
5. Go ¨rlich D, Prehn S, Hartmann E et al (1992) A
mammalian homolog of SEC61p and SECYp is
48
Jingze J. Wu and Alexander F. Palazzo
processed for immunofluorescence, we recommend that they
be extensively washed with PBS to fully remove any formamide, which can reduce the efficiency of immunolabeling. For
the immunostaining, it is best to use purified antibodies and to
avoid whole-serum preparations as these tend to contain
RNase which will reduce the smFISH signal. Many protocols
use whole serum to reduce background staining; however we
have found that this can be done by including 1% bovine serum
albumin and 0.1% Triton-X100 to the primary and secondary
antibody solutions.
24. We use Nikon Imaging Software (NIS-Elements) to collect and
process the micrographs.
25. To ensure that the fluorescence intensities between fields are
comparable, the exposure time should remain constant
between different fields of view and different coverslips. Furthermore, all of the imaging of one experimental set should be
done in one sitting to minimize any day-to-day fluctuations in
the intensity of the excitation light source.
26. To identify spots, use the Spot Detection Module on
NIS-Elements (Fig. 3) using the Bright, Clustered Method
option. For settings, for the “Typical Diameter” we use
0.32 μm, and for the “Remove Dim Objects” we set the level
so that few to no spots are selected in images taken in parallel of
cells where we either omit the smFISH probes or stain with
scrambled probes (see Note 19). This is essential as there is
always a low level of staining caused by autofluorescence or
probes binding to suboptimal targets. The total number of
spots in whole cell and nuclear ROIs are counted automatically
using the “Automated Measurement Results” and the number
of spots in the cytoplasm is calculated by subtracting the
nuclear spots from the total cell spots. The number of nuclear
foci can be used as a control to ensure that the unextracted and
digitonin-extracted cells have been stained equally well. Nuclei
should be impervious to digitonin extraction [20].
References
1. Cui XA, Palazzo AF (2014) Localization of
mRNAs to the endoplasmic reticulum. Wiley
Interdiscip Rev RNA 5:481–492
2. Rapoport TA (2007) Protein translocation
across the eukaryotic endoplasmic reticulum
and bacterial plasma membranes. Nature
450:663–669
3. Walter P, Ibrahimi I, Blobel G (1981) Translocation of proteins across the endoplasmic
reticulum. I. Signal recognition protein (SRP)
binds to in-vitro-assembled polysomes synthesizing secretory protein. J Cell Biol
91:545–550
4. Gilmore R, Blobel G, Walter P (1982) Protein
translocation
across
the
endoplasmic
reticulum. I. Detection in the microsomal
membrane of a receptor for the signal recognition particle. J Cell Biol 95:463–469
5. Go ¨rlich D, Prehn S, Hartmann E et al (1992) A
mammalian homolog of SEC61p and SECYp is
48
Jingze J. Wu and Alexander F. Palazzo