4 In Situ Nick End-Labeling: Electron Microscopical
35
Procedure
Preparation of samples
1. Cut the specimens into small pieces (5 X 5 X 5 mm in maximal
size).
2. Fix with the fixative for 2 hrs at 4°C.
3. Rinse and immerse in PB overnight at 4 DC.
4. Post-fix with osmium tetroxide for 2 hrs at room temperature
(RT).
5. Immerse in 0.1 M PB for 5 mins, three times.
6. Dehydrate in a series of graded ethanol solutions for 15 mins,
respectively.
7. Clear in propylene oxide for 15 mins, twice.
8. Embed in epoxy resin.
9. Make ultra-thin sections (more than 80 nm thick) by an ultramicrotome and mount on nickel grids.
ISELfTEM technique
Several buffers for the ISELITEM technique shown in the Materials Section are cited from the method of TdT-mediated dUTPbiotin nick end labeling (Gavrieli et aI1992). We recommend the
use of an available commercial non-radioactive apoptosis detection kit (e.g., Apop Tag™ : Oncor, Gaithersburg, MD, USA) to
obtain constant and trustworthly data, although the purchase of
the kit may be expensive. Therefore, we will state here the procedure for the ISELITEM technique, using Apop Tag™ kit (see
Fig. 2).
Each reaction is performed on droplets of medium placed on
Parafilm; the face of the grid with ultra-thin sections is floated,
incubated or washed on the droplets of the medium (Fig. 3).
Know how to use locking forceps, anticapillary forceps and
wire loop. We use locking forceps to securely grip the grid, while
washing in the beaker. Anti-capillary forceps are used when the
grid is transported between the droplets consisting the different
35
Procedure
Preparation of samples
1. Cut the specimens into small pieces (5 X 5 X 5 mm in maximal
size).
2. Fix with the fixative for 2 hrs at 4°C.
3. Rinse and immerse in PB overnight at 4 DC.
4. Post-fix with osmium tetroxide for 2 hrs at room temperature
(RT).
5. Immerse in 0.1 M PB for 5 mins, three times.
6. Dehydrate in a series of graded ethanol solutions for 15 mins,
respectively.
7. Clear in propylene oxide for 15 mins, twice.
8. Embed in epoxy resin.
9. Make ultra-thin sections (more than 80 nm thick) by an ultramicrotome and mount on nickel grids.
ISELfTEM technique
Several buffers for the ISELITEM technique shown in the Materials Section are cited from the method of TdT-mediated dUTPbiotin nick end labeling (Gavrieli et aI1992). We recommend the
use of an available commercial non-radioactive apoptosis detection kit (e.g., Apop Tag™ : Oncor, Gaithersburg, MD, USA) to
obtain constant and trustworthly data, although the purchase of
the kit may be expensive. Therefore, we will state here the procedure for the ISELITEM technique, using Apop Tag™ kit (see
Fig. 2).
Each reaction is performed on droplets of medium placed on
Parafilm; the face of the grid with ultra-thin sections is floated,
incubated or washed on the droplets of the medium (Fig. 3).
Know how to use locking forceps, anticapillary forceps and
wire loop. We use locking forceps to securely grip the grid, while
washing in the beaker. Anti-capillary forceps are used when the
grid is transported between the droplets consisting the different