prechilled at 4
C. Swirl gently to ensure proper washing and
aspirate wash buffer from the wells using a serological pipette
(see Note 13).
3. Gently add 2 mL of 25% EtOH to the side of each well. Tilt the
plate gently to ensure proper mixing and incubate at room
temperature for 15 min.
4. Remove the 25% EtOH solution from wells and gently add in
40% EtOH. Repeat for each subsequent EtOH concentration
until 100% (see Note 14).
5. Use forceps to remove coverslips from the well and place face
up on lint-free tissue to remove excess liquid.
6. Air dry the coverslips for 5 min at room temperature and
transfer them to a desiccator or a container with silica beads.
7. Mount coverslips on the SEM stage using double-sided carbon
tape and sputter coat appropriately for viewing under the
microscope.
3.5 Sample
Preparation for
Confocal Microscopy
1. Following invasion (see Subheading 3.3), add 3 mL of complete cell culture media containing 80 μg/mL of gentamicin to
each well. Incubate at 37
C in a CO 2 incubator for 1 h (see
Note 15).
2. Aspirate media containing gentamicin using a serological
pipette and replace with 3 mL complete culture medium containing 20 μg/mL gentamicin to each well. Incubate until the
desired timepoint (see Note 15).
3. Aspirate culture medium and wash the cells three times with
3 mL of 1Â PBS per well. Gently swirl the plates to ensure
proper washing (see Note 16).
4. Add 3 mL of fixing solution and incubate at 4
C overnight.
(see Note 12).
5. Aspirate fixing solution and rinse wells with 3 mL of 1Â PBS
chilled to 4
C (see Note 17).
6. Add 1 mL of fresh 1Â PBS to each well. Using a micropipette,
add acridine orange to each well. Swirl the plates gently to mix
and store at 4
C for 10 min.
7. Remove fluorescent stain by aspiration using a serological
pipette. Gently rinse each well three times with 1Â PBS (see
Note 18).
8. Using forceps, remove microscope slides from each well and
place face-up on lint-free tissue to dry for 10 min at room
temperature.
9. Invert and mount coverslips onto glass slides for viewing under
the microscope (see Note 19).
36
Andrew N. Osahor et al.
C. Swirl gently to ensure proper washing and
aspirate wash buffer from the wells using a serological pipette
(see Note 13).
3. Gently add 2 mL of 25% EtOH to the side of each well. Tilt the
plate gently to ensure proper mixing and incubate at room
temperature for 15 min.
4. Remove the 25% EtOH solution from wells and gently add in
40% EtOH. Repeat for each subsequent EtOH concentration
until 100% (see Note 14).
5. Use forceps to remove coverslips from the well and place face
up on lint-free tissue to remove excess liquid.
6. Air dry the coverslips for 5 min at room temperature and
transfer them to a desiccator or a container with silica beads.
7. Mount coverslips on the SEM stage using double-sided carbon
tape and sputter coat appropriately for viewing under the
microscope.
3.5 Sample
Preparation for
Confocal Microscopy
1. Following invasion (see Subheading 3.3), add 3 mL of complete cell culture media containing 80 μg/mL of gentamicin to
each well. Incubate at 37
C in a CO 2 incubator for 1 h (see
Note 15).
2. Aspirate media containing gentamicin using a serological
pipette and replace with 3 mL complete culture medium containing 20 μg/mL gentamicin to each well. Incubate until the
desired timepoint (see Note 15).
3. Aspirate culture medium and wash the cells three times with
3 mL of 1Â PBS per well. Gently swirl the plates to ensure
proper washing (see Note 16).
4. Add 3 mL of fixing solution and incubate at 4
C overnight.
(see Note 12).
5. Aspirate fixing solution and rinse wells with 3 mL of 1Â PBS
chilled to 4
C (see Note 17).
6. Add 1 mL of fresh 1Â PBS to each well. Using a micropipette,
add acridine orange to each well. Swirl the plates gently to mix
and store at 4
C for 10 min.
7. Remove fluorescent stain by aspiration using a serological
pipette. Gently rinse each well three times with 1Â PBS (see
Note 18).
8. Using forceps, remove microscope slides from each well and
place face-up on lint-free tissue to dry for 10 min at room
temperature.
9. Invert and mount coverslips onto glass slides for viewing under
the microscope (see Note 19).
36
Andrew N. Osahor et al.