3. Process the imaging data with imaging software (see Note 24).
4. Select regions of interest (ROI) for the whole cell and nucleus,
and then identify and count the fluorescent spots. The quantification of foci can then be used to determine the relative
association of any given Stellaris probe marked mRNA to the
ER (see Note 26).
4 Notes
1. To ensure sterility, add the fetal bovine serum and penicillin/
streptomycin to the DMEM in a biosafety cabinet. Since we
found that using cold DMEM can cause some U2OS cells to
detach during cell maintenance, we warm the DMEM in a
37
C water bath for at least 15 min prior to usage.
2. It is very helpful to use cell culture lines that have a very welldefined ER morphology. To that extent, we primarily use
human osteosarcoma cells (U2OS) and green African monkey
kidney fibroblast-like cells (COS7) in our experiments.
3. Sterilize by flaming the tips of the forceps for 2–5 s prior to use.
Ensure that the tips are not too hot when interacting with
coverslips, so as not to damage coverslips/cells.
4. Treating coverslips with acid helps to etch and clean the surface, which in turn helps promote cell spreading. Well-spread
cells permeabilize much more efficiently than rounded cells for
reasons that are still not entirely clear. Post-acid treatment,
ample washes with deionized H 2 O are necessary to remove
the acid completely and thus prevent unwanted acid contamination in later steps. UV treatment prior to storage helps to
sterilize the coverslips.
5. Warm up 1Â CHO buffer in 37
C water bath prior to use.
Typically, about 10–25 mL of 1Â CHO buffer is used per
experiment which could consist of two to a dozen 12 mm
coverslips. CHO buffer was formulated to maintain the integrity of ER preparations isolated from cell lysates [22].
6. We found that using ice-cold methanol extraction prior to
coverslip mounting helps reduce autofluorescent signals during
imaging.
7. Since we typically dilute the Stellaris probes 1:50 in deionized
H 2 O, and about 100 μL of the final diluted probe solution is
used per 12 mm coverslip, 1 mL of FISH hybridization buffer
is generally used in an experiment consisting of ten 12 mm
coverslips. Use this as a general rule of thumb as to how many
1 mL aliquots of FISH buffer to thaw out, so as to avoid
refreezing and rethawing.
44
Jingze J. Wu and Alexander F. Palazzo
4. Select regions of interest (ROI) for the whole cell and nucleus,
and then identify and count the fluorescent spots. The quantification of foci can then be used to determine the relative
association of any given Stellaris probe marked mRNA to the
ER (see Note 26).
4 Notes
1. To ensure sterility, add the fetal bovine serum and penicillin/
streptomycin to the DMEM in a biosafety cabinet. Since we
found that using cold DMEM can cause some U2OS cells to
detach during cell maintenance, we warm the DMEM in a
37
C water bath for at least 15 min prior to usage.
2. It is very helpful to use cell culture lines that have a very welldefined ER morphology. To that extent, we primarily use
human osteosarcoma cells (U2OS) and green African monkey
kidney fibroblast-like cells (COS7) in our experiments.
3. Sterilize by flaming the tips of the forceps for 2–5 s prior to use.
Ensure that the tips are not too hot when interacting with
coverslips, so as not to damage coverslips/cells.
4. Treating coverslips with acid helps to etch and clean the surface, which in turn helps promote cell spreading. Well-spread
cells permeabilize much more efficiently than rounded cells for
reasons that are still not entirely clear. Post-acid treatment,
ample washes with deionized H 2 O are necessary to remove
the acid completely and thus prevent unwanted acid contamination in later steps. UV treatment prior to storage helps to
sterilize the coverslips.
5. Warm up 1Â CHO buffer in 37
C water bath prior to use.
Typically, about 10–25 mL of 1Â CHO buffer is used per
experiment which could consist of two to a dozen 12 mm
coverslips. CHO buffer was formulated to maintain the integrity of ER preparations isolated from cell lysates [22].
6. We found that using ice-cold methanol extraction prior to
coverslip mounting helps reduce autofluorescent signals during
imaging.
7. Since we typically dilute the Stellaris probes 1:50 in deionized
H 2 O, and about 100 μL of the final diluted probe solution is
used per 12 mm coverslip, 1 mL of FISH hybridization buffer
is generally used in an experiment consisting of ten 12 mm
coverslips. Use this as a general rule of thumb as to how many
1 mL aliquots of FISH buffer to thaw out, so as to avoid
refreezing and rethawing.
44
Jingze J. Wu and Alexander F. Palazzo