Measurement of Autophagy in Cells and Tissues - Protein Misfolding and Cellular Stress in Disease and Aging - page 201

11. The membrane was washed at least five times, for 10 min each
time, in TTBS at room temperature.
12. The reaction of horseradish peroxidase with chemilumines-
cent reagent was started on the membrane, and the signal was
detected on X-ray film or using a CCD camera (Fig. 3 ).

3.4. Immunofluore-
scence Analysis
for Autophagosomes
and Autolysosomes

1. After washing in TBS, cells were incubated in cell-fixative
solution at 37°C for 5 min.
2. The cells were rinsed twice in TBS.
3. Cells were permeabilized in digitonin solution at 37°C for
5 min. The solution was then discarded by aspiration.
4. Excess digitonin was quenched by incubation in quenching
solution at 37°C for 5 min. The solution was then discarded
by aspiration.
5. The cells were rinsed twice in TBS.
6. Cells were incubated in a drop of Image-IT FX at 37°C for
30 min.
7. The cells were rinsed three times in TBS.
8. Cells were incubated in IF-primary antibody solution at 37°C
for 60 min.
9. The cells were washed three times in TBS for 5 min each time
at 37°C.
10. Cells were incubated in IF-secondary antibody solution at
37°C for 60 min.
11. The cells were washed five times in TBS for 5 min each time
at 37°C.
12. The cells were mounted on glass slides in mounting
medium.
13. Fluorescent images were obtained using a fluorescence micro-
scope (Fig. 4 ).

3.5. Conventional
Electron Microscopy
for Cultured Cells

1. Cells were grown on round coverslips ~12 mm in diameter
(see Note 10 for tissue preparations ) .
2. Immediately after removing the medium, 2% PFA-2% GA
buffered with 0.1 M PB, pH 7.4 was added (see Note 11).
3. Cells were washed three times with 7.5% sucrose in PB.
4. Preparations were postfixed with OsO 4 -ixative at 4°C for
30 min in dark chamber (e.g. refrigerator).
5. Preparations were then washed three times with pure water.
6. Dehydration was performed by successive immersion in 50%,
70%, 80%, and 95% ethanol for 5-10 min.
7. Coverslips were transferred into small glass dishes containing
100% ethanol, immersed for 10 min, and the ethanol was