97- Wash the wells three times with 200 μl of PBST.
- Add ECL™ Anti-rabbit IgG Horseradish Peroxidase linked
F(ab)′ 2 fragment (GE Healthcare, UK) in PBST and 1:20
diluted ImmunoBlock (total volume 10 ml) and incubated for
1 h at 37 °C. - Wash the wells three times with 200 μl of PBST.
- Develop color using TMB Peroxidase EIA Substrate Kit
according to manufacturer’s instructions. - Stop reaction by adding 0.1 M sulfuric acid and read absor-
bance at 450 nm ( see Fig. 2 ). - Fix skin tissues in acetone (the Amex method) to better pre-
serve antigens as well as high molecular weight DNA and RNA
that are normally destroyed by the routine formalin fi xation
and paraffi n-embedding process [ 13 ] ( see Note 6 ). - Briefl y, immerse tissues in acetone at 4 °C, transfer to a freezer
at −20 °C, and fi x overnight. - Dehydrate tissues in acetone, fi rst at 4 °C for 15 min and then
for another 15 min at room temperature. - Clear tissues twice in methyl benzoate for 15 min, and then
twice in xylene for 15 min at room temperature. - Embed in paraffi n at 60 °C for 2–3 h in a vacuum-evaporating
embedder. - Prepare thin sections using microtome and deparaffi nize with
xylene for 30 min. - Immerse in acetone 2–3 times.
- Wash with PBS three times for 10 min.
- Block sections with 10 % goat serum for 1 h at room
temperature. - Wash sections with PBS for 5 min and incubate with appropri-
ate primary antibody for 1 h at room temperature or at 4 °C
overnight. Double staining can be done using anti-caspase-
14 mAb and cleavage-site-directed antibody, h14D146 or
h14Y178. - Wash with PBS three times for 10 min.
- Incubate with secondary antibodies (e.g., Alexa Fluor 555 or
488; Molecular Probes) for 1 h at room temperature. - Wash with PBS three times for 10 min.
- Immerse sections in VECTASHIELD and stain with DAPI
( see Fig. 3 ; see Note 7 ).
3.7 Immunohisto-
chemical Localization
of Caspase-14
Caspase-14 Methods