3. 10% Formamide wash buffer: 2Â SSC, 10% formamide. Make
up to 1 L. Store at room temperature.
4. Stellaris mRNA probes (LGC Biosearch Technologies): Dissolve Stellaris mRNA lyophilized probes in RNase-free water to
a final concentration of 12.5 μM and store at À20
C (see Notes
8 and 9) in a dark tube (see Note 10).
5. smFISH staining dish: Moisten the base of a 150 mm petri dish
with deionized water, and then place a piece of parafilm cut to
the size of the dish on top of the wet surface. Smooth out any
excess water and bubbles underneath the parafilm using
RNase-free gloves, so that the surface is flat (similar to the
heat block parafilm preparation, refer to Subheading 2.2,
item 4).
6. Mounting solution with DAPI (see Note 11).
3 Methods
3.1 Digitonin
Extraction
1. Seed cells on 12 mm acid-treated coverslips in 12-well cell
culture plates at least 1 day prior to the experiment (see
Note 12).
2. Pipette 100 μL of 0.025% digitonin extraction solution onto
the parafilm on top of the 40
C heat block. It should form a
firm liquid droplet. Repeat this process until the number of
drops equals the number of coverslips to be extracted. This
should be done immediately before you plan to extract and fix
the cells.
3. To two 35 mm dishes, add 3 mL of warm (37
C) CHO
buffer each.
4. To a 12-well plate, add 1 mL of 4% paraformaldehyde fixing
solution per well per coverslip (adjust as needed). Place these
near the heat block. For example, if there are eight coverslips in
the experiment, add 1 mL of 4% paraformaldehyde fixing solution to 8 wells of a 12-well plate, and place aside to facilitate
quick fixation later.
5. Remove the cells from the 37
C incubator and place them near
the heat block.
6. Using a pair of jeweler’s forceps, remove a coverslip from the
12-well and dip it into the two 35 mm dishes with CHO buffer
(see step 3) consecutively to wash off excess media.
7. Quickly blot off excess liquid from the coverslip by touching
the edge of the coverslip to a Kimwipe sheet (see Note 13), and
then place the coverslip on a droplet of digitonin extraction
buffer (see step 2) on the heat block, cell side down. When
Visualizing ER-Associated mRNA
41
up to 1 L. Store at room temperature.
4. Stellaris mRNA probes (LGC Biosearch Technologies): Dissolve Stellaris mRNA lyophilized probes in RNase-free water to
a final concentration of 12.5 μM and store at À20
C (see Notes
8 and 9) in a dark tube (see Note 10).
5. smFISH staining dish: Moisten the base of a 150 mm petri dish
with deionized water, and then place a piece of parafilm cut to
the size of the dish on top of the wet surface. Smooth out any
excess water and bubbles underneath the parafilm using
RNase-free gloves, so that the surface is flat (similar to the
heat block parafilm preparation, refer to Subheading 2.2,
item 4).
6. Mounting solution with DAPI (see Note 11).
3 Methods
3.1 Digitonin
Extraction
1. Seed cells on 12 mm acid-treated coverslips in 12-well cell
culture plates at least 1 day prior to the experiment (see
Note 12).
2. Pipette 100 μL of 0.025% digitonin extraction solution onto
the parafilm on top of the 40
C heat block. It should form a
firm liquid droplet. Repeat this process until the number of
drops equals the number of coverslips to be extracted. This
should be done immediately before you plan to extract and fix
the cells.
3. To two 35 mm dishes, add 3 mL of warm (37
C) CHO
buffer each.
4. To a 12-well plate, add 1 mL of 4% paraformaldehyde fixing
solution per well per coverslip (adjust as needed). Place these
near the heat block. For example, if there are eight coverslips in
the experiment, add 1 mL of 4% paraformaldehyde fixing solution to 8 wells of a 12-well plate, and place aside to facilitate
quick fixation later.
5. Remove the cells from the 37
C incubator and place them near
the heat block.
6. Using a pair of jeweler’s forceps, remove a coverslip from the
12-well and dip it into the two 35 mm dishes with CHO buffer
(see step 3) consecutively to wash off excess media.
7. Quickly blot off excess liquid from the coverslip by touching
the edge of the coverslip to a Kimwipe sheet (see Note 13), and
then place the coverslip on a droplet of digitonin extraction
buffer (see step 2) on the heat block, cell side down. When
Visualizing ER-Associated mRNA
41