Immunocytochemistry
Log-grown A549 cells were seeded on sterile coverslips, and the cells
were treated with two concentrations of PP-22 (0 or 4 μM) for 24 hours.
Cells on the coverslips were fixed with 4% paraformaldehyde (PFA) for
10 minutes. Cell washed 3 times with cold PBS every 3 minutes,
permeabilize cells with 300 μl of 0.25% Triton-X 100 at 4 ° C for 16
minutes, and treat cells with 4% bovine serum albumin soluble backgrou
binding for 1.5 hours, and continuously extend with LC3 antibody (1:
100) at 4 ° C. Cell is washed 3 times with PBST (PBS with Tween-20),
then keep at room temperature with Alexa Fluor 488 labeled goat
anti-rabbit IgG for 1.5 hours temperature according to manufacturer’s
instructions. Then, the cell stained with 4 ’, 6-dimidyl-2-phenylindole,
dihydrochloride (DAPI) (300 μl / group for 10 minutes), and washed 3
times with PBST again. Cell detected under a confocal microscope
(Olympus, Japan).