- Thereupon wash the coverslips two times with PBS and one
time with H 2 O(seeNote 9). - Add 500μl of Hoechst solution for 5 min for staining of the
cellular DNA (seeNote 9). - Perform two last washing steps using 1 ml of H 2 O(seeNote 9).
- To mount the coverslips and protect the fluorescence signal
from bleaching, spot 12μl of Mowiol solution onto a glass
microscope slide (seeNote 10). Usually up to two coverslips
can be placed next to each other on one microscope slide. Place
the coverslips onto the Mowiol spots with the cells facing the
solution, and incubate for 15 min at room temperature until
image acquisition or storage at 4C. - Acquire all images with the confocal laser-scanning microscope
using a 63x water immersion objective. Spot 50μl of ultrapure
water onto the objective before placing the microscope slide
on top. - Confocal laser-scanning microscopy allows simultaneous
acquisition of the Hoechst, Alexa Fluor 488, EGFP, and
mCherry fluorescent signal from a single focal plane. Excite
Hoechst with an UV laser and detect the emission signal
between 425 and 483 nm, while the Alexa Fluor 488 or
EGFP fluorophore should be exited with an Argon laser, and
the detection takes place between 493 and 550 nm. The exci-
tation of the fluorophore mCherry should be mediated by a
helium-neon laser and detected between 562 and 630 nm.
Always set the pinhole to 1 airy unit (AU), and choose a
resolution of 10241024 pixels for all pictures (seeNotes
36 – 38 ). - Perform the editing of the pictures with help of the confocal
laser-scanning microscope software (seeNote 37). - Quantify the LC3 dots per cell with the ImageJ software. Set
the brightness and contrast of the images to isolate the dots
and eradicate the background staining. Further analysis
requires conversion to an 8-bit binary image. To count the
thresholded dots, apply the Analyze Particles algorithm, and
set the minimum size and maximal pixel area size>0.1μm(see
Notes 37– 39 ).
4 Notes
- Protease and phosphatase inhibitors have to be added freshly to
the lysis buffer of every experiment where phosphorylation
should be assessed. Suggested phosphatase inhibitor stock
solutions: 1 Mβ-glycerophosphate in H 2 O. 100 mM vanadate
(Na 3 VO 4 )inH 2 O. 100μM okadaic acid in DMSO. Store all
solutions in aliquots at 20 C.
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