Biology Reference
In-Depth Information
binding: an unintended primary antibody, another fluorochrome-labeled secondary
antibody, or directly to fixed cells. To test for such unintended signals (as controls for
Fig. 12.1
), we stained two coverslips, omitting one of the primary antibodies in each
case—the anti-FLAG from the first coverslip and the anti-perilipin 3 antibody from
the second coverslip. Both coverslips were exposed to both secondary antibodies.
The first coverslip showed no appreciable signal in the green channel with imageable
signal in the red channel, and the second coverslip showed no appreciable signal in
the red channel with imageable signal in the green channel. This demonstrated that
the channels are independently imageable. However, these controls do not address
the specificity of the primary antibodies.
12.2.7
Antibody staining protocol
1.
Grow and treat cells on coverslips in six-well plates.
2.
Aspirate media. Do not wash cells.
3.
Add 2 ml of fix solution to each well (cells will be facing up).
1
4.
Incubate 10-20 min at room temperature then rinse coverslips four times with
2 ml PBS.
5.
Replace PBS with 2 ml microscopy buffer. (After fixation cells can be stored at
4
C in microscopy buffer for up to 4 weeks.)
6.
Place coverslips cell-bearing side up
2
on a parafilm-lined tray with a cover
(
Fig. 12.2
A). To avoid drying place a small water saturated paper towel inside
the tray.
7.
Add primary antibodies diluted in 150
l microscopy buffer to the cell-bearing
side of coverslips (
Fig. 12.2
B) and cover with lid.
8.
After 1 h, rinse coverslips four times with PBS and apply fluorochrome-antibody
conjugates (secondary antibodies) in microscopy buffer and cover with lid.
9.
After 1-h incubation in the dark, rinse coverslips five times with PBS.
10.
Pipette a bead of elvanol (or similar mounting solution) about 2 mm from the
top edge of a microscope slide.
11.
Dip coverslip in distilled water to wash off PBS.
12.
Wipe water off the noncell-bearing side of the coverslip (
Fig. 12.2
C).
13.
Mount coverslip on slide so that cells are sandwiched between the two pieces of
glass. Place the edge of the coverslip between the top edge of the slide and the
bead of elvanol. Then lower slowly until the coverslip is flat (
Fig. 12.2
D).
14.
Do not get elvanol on noncell-side of the coverslip.
m
1
In some cases formaldehyde fixation abolishes antibody binding. In this case, fix cells by adding 2 ml
100% methanol at
20
C, and incubate at that temperature for 4 min. Wash five times in PBS, then
proceed as per protocol (step 5). Fat droplet morphology will be dramatically compromised (
DiDonato
& Brasaemle, 2003
), but may still be informative.
2
Cells on 22-mm square coverslips can be stained with less than 50
l of antibody by placing the cov-
erslip cell-bearing side down on a drop of antibody. The volume of antibody used can be further re-
duced by using smaller coverslips if necessary.
m
