Biomedical Engineering Reference
In-Depth Information
11. Permeabilize the cells with 0.2 % Triton X-100 in PBS for
5 min. Remove the permeabilization buffer and wash cells
twice with PBS.
12. Incubate the cells in blocking buffer for 15 min.
13. Prepare primary antibodies by diluting the chicken anti-Myc
antibody at 1:1,000 in monoclonal mouse anti-cation-
independent MPR antibody culture supernatant.
14. Add the diluted primary antibodies on top of each coverslip
and incubate at room temperature for 1 h (
see
Note 22
). Make
sure the antibody solution covers the entire surface of the
coverslips.
15. Remove primary antibodies and wash coverslips three times for
5 min each with wash buffer on a Belly Dancer or other plat-
form shaker.
16. Prepare secondary antibodies by diluting the Alexa Fluor 488
goat anti-mouse antibody 1:2,000 and the Alexa Fluor 555
goat anti-chicken antibody 1:2,000 in wash buffer.
17. Add secondary antibodies on top of each coverslip and incu-
bate at room temperature for 1 h (
see
Note 22
).
18. Remove the secondary antibodies and wash the coverslips two
times for 5 min each with wash buffer and once with PBS for
5 min on a Belly Dancer.
19. Clean glass slides and put a drop of Mowiol on top.
20. Transfer 500 ml of distilled water into a beaker. Dip the cover-
slip into distilled water for 10 s; absorb any residual water
droplets by placing the edge of the coverslip onto a piece of
Kimwipe or other tissue.
21. Carefully put the coverslip (cells facing down) on top of the
Mowiol droplet; avoid bubbles and clean away excessive
Mowiol using a Kimwipe. Let the glass slides dry in the dark
overnight at room temperature before imaging. Glass slides
can be transferred to 4 °C in the dark for long-term storage.
22. Acquire images using a fl uorescence microscope at 60× [
13
]:
(a) Acquire 3-5 Z-sections with a
Z
-axis drive (MFC-2000,
Applied Scientifi c Instrumentation, Eugene, OR) in 0.2-
mm steps.
(b) Use software to deconvolve images using a theoretical
point spread function.
23. Score rescue experiment images. Rescued cells display a more
compact perinuclear staining for MPRs, whereas cells depleted
of the tethering protein or non-rescued cells show a more dis-
persed punctate staining that extends to the cell periphery
(Fig.
2
) (
see
Note 23
).
