Before application of the probe, frozen sections were warmed to room
temperature, and paraffin sections were deparaffinized. Because tissue
to be processed for JB-4 required the hybridization step before JB4
embedding, it was treated similarly to the frozen and paraffin sections
at this point. All sections and tissue wedges were then incubated in
0.2 N HCl, treated with proteinase K, washed in phosphate-buffered
saline, immersed 0.5% acetic anhydride in 0.1 M triethanolamine at pH
8, prehybridized in mRNA hybridization solution (DAKO, Carpinteria,
CA), and hybridized in a solution containing 300 ng/ml of either the
sense or antisense probe. All sections and tissue wedges were incubated
in a closed moist chamber at 55°C overnight to allow hybridization.
After hybridization, microscopic slides and tissue wedges washed in
SSC, incubated in 4 μg/ml RNase A to remove unbound probe, and washed
in SSC at 50°C. All slides and wedges were then blocked with 5%
bovine serum albumin in 150 mM NaCl, 100 mM Tris–HCl (pH 7.50)
for 30 minutes at room temperature and incubated in the same
solution containing a 1:200 dilution of anti-DIG alkaline phosphatase
conjugate (Boehringer Mannheim) for 2 hours at 37°C. After washing in
150 mM NaCl, 100 mM Tris–HCl (pH 7.5), the nitroblue
tetracolium/5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP) color
detection reaction was then performed according to the manufacturer’s
instructions (Boehringer Mannheim). The color detection reaction was
monitored microscopically and interrupted after 2 hours by washing in
10 mM Tris, 1 mM EDTA. Sections were then mounted and coverslips
applied, whereas the tissue wedges were processed into JB-4 plastic and
sectioned at 5 μm thickness. Nuclear fast red was used as a
counterstain for some sections.