Measurement of Autophagy in Cells and Tissues - Protein Misfolding and Cellular Stress in Disease and Aging - page 202

exchanged with fresh 100% ethanol once and immersed for
10 min (see Note 12).
8. Immersion in propylene oxide twice for 5 min.
9. Immersion in a mixture of propylene oxide and Epon 812
(equal proportions) for 20 min.
10. Immersion in Epon 812 mixture overnight under weak nega-
tive pressure generated with a vacuum pump.
11. Embedded in fresh Epon 812 mixture.
12. Heated at 80°C for 2 days.
13. Coverslips were carefully removed while the Epon block was
heated appropriately.
14. The side with cultured cells was confirmed, and ultrathin sec-
tions (approximately 70 nm thickness) were cut using a
microtome (see Note 13).
15. Staining was performed with diluted uranyl acetate solution
for 5 min.
16. Washing with water.
17. Staining with 0.25-0.3% lead citrate for 5 min.
18. Observation under an electron microscope.
19. If necessary, the number and size of autophagosomes (see
Note 14) were determined using an imaging software.
4. Notes
1. E64d is a derivative of E64 and E64c. E64d is a membrane-
permeable analog of E64c, while E64 and E64c are mem-
brane-impermeable. Therefore, E64 and E64c have little
inhibitory effect on cathepsins compared with E64d when
added to culture medium. Leupeptin (Peptide Institute) is
intraperitoneally injected into rats (or mice) when autolyso-
somes are prepared from the liver, as leupeptin effectively
accumulates in the liver probably via endocytosis. As leupep-
tin is membrane-impermeable, care should be taken to deter-
mine whether leupeptin inhibits the activity of cathepsin B
when used to treat cells and other tissues. Leupeptin is dis-
solved at 1 mg/mL in H 2 O. The 1,000× concentrated stock
solution is stored at -20°C, at which temperature it is stable
for at least one month.
2. Storage of pepstatin A for over 3 months should be avoided.
When pepstatin A is added to the medium, it should be mixed
quickly, as it tends to precipitate.