Biomedical Engineering Reference
In-Depth Information
To dishes washed with EBSS, add:
Starved: add 1 ml EBSS.
Starved plus BafA: EBSS plus 100 nM bafi lomycin A1.
Return to incubator and incubate for 90-120 min.
See
Note 4
for alternative treatments to induce or inhibit
autophagy.
5. Wash cells on glass coverslips once with 2 ml PBS.
Fix in 3 % PFA in CMF-PBS for 20 min (
see
Note 5
).
6. Wash twice with PBS, and quench aldehydes with 50 mM
NH
4
Cl in PBS for 10 min.
7. Wash twice with PBS, and permeabilize with 0.2 % Triton
X-100 in PBS for 3 min maximum (
see
Note 5
for alternative
permeabilization protocols).
8. Wash 3 times with PBS, replace fi nal wash with PBS, 0.2 %
gelatin, and incubate for 20 min.
9. Set up a moist chamber for the antibody incubation. We sug-
gest lining the lid of a plastic box with wet fi lter paper and
covering it with parafi lm. The bottom of the box would be
placed down on the lid to create the chamber (resulting in the
plastic box sitting upside down on its lid). Number or divide
the area of the parafi lm to correspond to the number of cover-
slips using a fi ne-tipped marker.
10. Prepare the primary antibodies. Make a cocktail of all 3 pri-
mary antibodies in PBS-gelatin (
see
Note 6
) if primary anti-
bodies are compatible. Prepare 80
ʼ
L per coverslip.
11. Drop 80
L of primary antibody on parafi lm. Remove the cov-
erslip from well with fi ne-point forceps. Drain excess liquid
from coverslip by touching on a piece of absorbent tissue.
Place coverslip cell-side down on drop of antibody. Incubate
1 h at room temperature or 4 °C overnight. Ensure coverslips
do not dry out while fl oating on the drop of antibody.
12. Remove the coverslip from drop using fi ne-tipped tweezers,
and place back into 12-well plate with cell-side facing up.
13. Wash 3 times with PBS-gelatin.
14. Dilute fl uorescent secondary antibodies in PBS-gelatin.
See
Note 6
. Use fl uorescent antibodies validated in laboratory.
Use 80
ʼ
L per coverslip.
15. Replace parafi lm in chamber and remark. Repeat
step 11
.
16. Repeat
step 12
. Wash twice with PBS-gelatin and then once
with PBS.
17. Prepare glass slides. Mark frosted region with pencil. Drop
about 20
ʼ
ʼ
L Mowiol mounting medium onto slide. It is
