- Thereafter wash the blots with TBS-T three times for at least
5 min each on a horizontal shaker. - Afterward incubate the blot with the according secondary
antibody diluted in TBS-T, 5% (w/v) skim milk powder, for
1 h at room temperature on a rocking device. - Following the incubation step, wash the blot again with TBS-T
three times for at least 10 min each on a horizontal shaker. - To develop the blot mix the ECL substrate solutions (Lumi-
nol/Enhancer and Stable Peroxide Buffer) in a 1:1 ratio in a
reaction tube immediately before usage (seeNote 31). - Place the blot on the metal panel of the chemiluminescence
imager, cover with the fresh ECL solution mixture, and place
inside the imager to take a chemiluminescence picture (see
Note 31). - Reuse the blot to detect additional proteins with differing
molecular weights. To do so, wash the blot with TBS-T several
times to remove the residual ECL solution, and afterward
repeat the procedure fromstep 7(seeNote 32). - To evaluate and quantify detected protein amounts on the
immunoblot, use the ImageJ software for densitometry
according to Chapter 30.13 of the ImageJ user guide (see
Notes 3and 33 ).
3.6
Immunofluorescence
Staining of Adherent
Cells and Confocal
Microscopy
- After the cell incubation (seeSubheading3.4 ,step 1), transfer
coverslips to a 12-well dish with the cells facing up (seeNote 34). - Fixate the cells by incubation with 500μl of fixation solution
per well for 20 min. Remove residual PFA by a washing step
with 1 ml of PBS. - Thereafter treat the fixated cells with 1 ml of permeabilization
solution for 15 min to permeabilize their membranes (see
Notes 7and 8 ). - To reduce unspecific binding of the antibodies, block the cov-
erslips with 1 ml of IF blocking solution for 30 min. - Dilute the primary anti-LC3 antibody 1:50 in IF antibody
diluent solution, and spot 50μl per coverslip onto a Parafilm.
Place the coverslips on the antibody spots with the cells facing
the solution, and incubate in a humid box for 1 h at 37C(see
Note 35). - Perform the subsequent three washing steps in the 12-well dish
again using 1 ml of IF antibody diluent solution. - The incubation with the secondary fluorophore-coupled anti-
body takes place as described for the primary antibody. Use
60 μl of the antibody dilution per coverslip, and keep in the
dark for all subsequent steps or at least keep the light exposure
to a minimum.
384 Sarah Krieg et al.