Caspases,Paracaspases, and Metacaspases Methods and Protocols

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4 Notes

1. For the measurement of revC14-Y178, sodium citrate can be
   omitted.


2. Caspase-14 requires high concentration of kosmotropic salt
   for its activation. Usually 1–1.3 M sodium citrate is used since
   it shows neutral pH in the solution.


3. Substrates WEHD-AFC or WEHD-MCA are most suitable for
   caspase-14 activity measurement. Although substrate specifi c-
   ity of caspase-14 resembles that of caspase-1, YVAD-AFC
   (MCA) is not recommended, since it shows inhibitory effect.


4. It is quite hard to obtain purifi ed caspase-14 from corneocyte
   extracts for the characterization because of very elaborate puri-
   fi cation procedures. Chymotrypsin-activated caspase-14 or
   constitutively active revC14 should be used as an alternative
   source.


5. Activity of KLK7 is strongly suppressed in the presence of high
   concentrations of kosmotropic salts. For the activation assay, it
   is necessary to perform two-step procedure in different buffer
   systems.


6. When using the Amex method, preservation of antigenicity is
   almost equivalent to that of frozen sections. Thin sections of
   paraffi n-embedded samples can be kept at 4 °C for 1 year.


7. Color development can be done using the 3,3′-diaminobenzi-
   dine (DAB) method, which produces an easily detectable
   brown color. The advantage of this method is that it enables
   permanent mounting of the sections. The disadvantage of the
   method is that it precludes double staining.

Fig. 3 Immunohistochemical localization of procaspase-14 and mature caspase-14. Procaspase-14 stained
with H99 was present from spinous to granular layers. In contrast, the mature caspase-14, which was detected
with h14D146, showed confi ned localization, being only found in the cornifi ed layer

Caspase-14 Methods