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should be examined. The time of incubation and the amount
of substrate concentration should be used for the assay within
the linear range.- Cleavage of initiator caspases is not necessary for their activa-
tion. The detection of proteolytic fragments due to internal
proteolysis of these caspases may therefore, not represent true
activation of the initiator caspases. Caspase-8 and -9 may be
active without being cleaved or without altered mobility on
western blots. Thus, additional methods including enzymatic
activity assay should be performed. - Caspase-cleaved products in a mouse tissue may not remain
stable and may become targets of other proteases for further
proteolysis. Therefore, the level of cleaved product sometimes
may not refl ect true caspase activity. - The method relies on the source and availability of high quality
antibodies for the pro-form and cleaved form of caspases. - Leaving samples longer than 3 days in fi xative (over-fi xation)
should be avoided as it can lead to uneven and weak staining. - In case of incomplete antigen retrieval the duration of micro-
waving can be extended (timing needs to be adjusted by user
as it depends on specimen preparation, thickness and fi xation,
and microwave power) or alternative methods like treatment
of deparaffi nized slides in a water bath or a steamer and enzy-
matic digestion can be applied. For a steamer or water bath,
preheat glass jar fi lled with antigen retrieval buffer until boiling
(temperature >95 °C), insert slides and continue heating for
20–40 min. Let glass jar cool to room temperature (~20 min).
Wash sections twice in PBS-T for 5 min and continue with the
staining procedure as described above. For enzymatic diges-
tion of deparaffi nized sections, transfer slides into a buffer con-
taining 50 mM Tris–HCl, pH 8.0, 5 mM EDTA, and 10 μg/ml
proteinase K and incubate at 37 °C for 5 min. Wash sections
twice in PBS-T for 5 min and continue with the staining pro-
cedure as described above. - To reduce nonspecifi c staining, BSA in both the “Blocking
buffer” and the “Antibody dilution buffer” can be substituted
with serum from the species in which the secondary antibody
was raised (e.g., goat serum instead of BSA in the specifi ed
buffers when using AlexaFluor-594 goat anti-rabbit antibody).
The use of detergents in the blocking buffer can be varied to
improve antigen detection (e.g., up to 0.1 % Triton X-100). - Primary antibodies directly conjugated to a fl uorescent
reporter, such as FITC, can be more effi cient in detection of
the antigen. FITC-conjugated rabbit polyclonal anti-active cas-
pase-3 antibody is available with Cell Signaling Technology Inc.
The secondary antibody is not required after incubation of tis-
sue section with fl uorochrome-conjugated primary antibodies.
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