162
- Glass Coverslips: 12 mm circles (fi t in 12 or 6-well culture
plates), Thickness No. 1. Coverslips should be cleaned with
ethanol and sterilized prior to use. - Phosphate-buffered saline (PBS), pH 7.4.
- Fixative, 4 % Formaldehyde: Prepare formaldehyde using either
paraformaldehyde powder solubilized in PBS or dilute 36.7 %
formaldehyde in PBS. Methanol-free formaldehyde is recom-
mended ( see Note 10 ). The solution should be used fresh. - Blocking/permeabilizing Buffer: 1–5 % bovine serum albumin
(BSA) and 0.3 % Triton™ X-100 in PBS ( see Note 11 ). - Antibody Dilution Buffer: 1 % BSA and 0.3 % Triton™ X-100
in PBS. - Primary antibodies:
Anti-cleaved caspase-3 (Asp175) pAb (Cell Signaling).
Anti-cleaved caspase-9 (Asp315) pAb (Thermo Scientifi c).
Anti-cleaved caspase-6 pAb (Millipore). - Fluorochrome-conjugated secondary antibodies: Alexa Fluor ®
488 (emission color-green) or Alexa Fluor ® 594 (emission
color-red) conjugated goat anti-rabbit IgG (H + L) (e.g., from
Life Technologies). - Nuclear counterstaining: Hoechst 33342 or DAPI. Prepare a
dilution of Hoechst 33342 or DAPI in PBS in a fi nal concen-
tration of 1 mg/mL. - VECTASHIELD ® Mounting Media (Vector Laboratories) or
equivalent. - Specimen glass slides.
- Nail polish.
Apart from primary Abs listed below, the materials are identical to
what is described in 2.1.1–2.1.4.- Anti-cleaved PARP (Asp214) Antibody (Cell Signaling).
- Anti-Bid antibody (Cell Signaling) ( see Note 13 ).
- Anti-cleaved Lamin A (Asp 230) antibody (Abcam).
- Phosphate-buffered saline (PBS), pH 7.4.
- Pure methanol.
2.2 Immuno-
cytochemical
Detection of Active
Caspases
2.2.1 Sample
Preparation
2.2.2 Immunostaining
2.3 Immunoblot
Analysis of Caspase
Substrate Cleavage
( See Note 12 )
2.4 Detection of
Cytokeratin 18
Cleavage by Flow
Cytometry
2.4.1 Sample
Preparation
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