2.8 Alkaline phosphatase staining
For ALP staining in cells, human or rat DP cells were seeded in 12-well
plate on coverslips. Cells were incubated with KY19382 for 48 hours and
washed twice with cold PBS and were fixed in formalin for 15 minutes at
room temperature. Then cells were incubated with nitro-blue tetrazolium
and 5-bromo-4-chloro-3’-indolyphosphate solution (NBT/BCIP solution,
Sigma Aldrich). The reaction was stopped by washing with PBS. Dark blue
staining was a positive signal for ALP.
For ALP staining of tissues, 20-μm cryosections were dried for 4 hours,
then fixed in 4% paraformaldehyde for 5 minutes. After being washed
with PBS, the sections were incubated in NBT/BCIP solution (NBT/BCIP
tablets, Roche Diagnostics, Rotkreuz, Switzerland) for 30 minutes. The
slides were counterstained with nuclear fast red solution (Sigma
Aldrich) for 30 seconds, washed in distilled water, and then dried for 2
hours. The slides were incubated in 100% xylene for 30 seconds, then
mounted using Permount.
For whole mount ALP staining, the wounded skin tissues were incubated 5
mM EDTA in PBS at 37 oC for 6 hours. The dermis was
separated under the microscope (Eclipse TE2000-U; Nikon). The dermis was
fixed in 4% paraformaldehyde for 10 minutes, washed with PBS and then
incubated in NBT/BCIP solution (NBT/BCIP tablets, Roche Diagnostics) for
10 minutes.