Caspases,Paracaspases, and Metacaspases Methods and Protocols

(Wang) #1

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  1. 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.

  2. Phosphate-buffered saline (PBS), pH 7.4.

  3. 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.

  4. Blocking/permeabilizing Buffer: 1–5 % bovine serum albumin
    (BSA) and 0.3 % Triton™ X-100 in PBS ( see Note 11 ).

  5. Antibody Dilution Buffer: 1 % BSA and 0.3 % Triton™ X-100
    in PBS.

  6. 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).

  7. 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).

  8. 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.

  9. VECTASHIELD ® Mounting Media (Vector Laboratories) or
    equivalent.

  10. Specimen glass slides.

  11. Nail polish.


Apart from primary Abs listed below, the materials are identical to
what is described in 2.1.1–2.1.4.


  1. Anti-cleaved PARP (Asp214) Antibody (Cell Signaling).

  2. Anti-Bid antibody (Cell Signaling) ( see Note 13 ).

  3. Anti-cleaved Lamin A (Asp 230) antibody (Abcam).

  4. Phosphate-buffered saline (PBS), pH 7.4.

  5. 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


Magnus Olsson and Boris Zhivotovsky

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