Frozen sections were allowed to defrost at room temperature for at
least 1 hour and hybridized with probe overnight at 65°C. Slides were
washed at 65°C in SSC buffer (3 M sodium chloride, 0.3 M sodium
citrate, pH 7) with 50% formamide and 0.1% Triton X-100 and then at
room temperature in TBST (0.14 M NaCl, 2.7 mM KCl, 0.025 M Tris-HCl, pH
7.5, 1% Triton X-100). Slides were then blocked in 10%
heat-inactivated sheep serum (in TBST) for >l hour at room
temperature. Anti-DIG AP-Fab fragment (in 10% heat-inactivated sheep
serum in TBST) was then added to each section and incubated overnight
in a humidified chamber at 4°C. Slides were washed in TBST at room
temperature and then in NTMT (100 mM NaCl, 100 mM Tris-HCl, pH 9.5, 50
mM MgCl2, 0.1% Triton X-100). Staining was
performed in the dark with nitroblue tetrazolium (NBT + 3.5%
5-bromo-4-chloro-3-indolyl phosphate [BCIP] in NTMT. After a few
hours, staining reaction was checked, though color development
could take up to 24 hours at 4°C. The reaction was stopped using
distilled water, and the sections fixed in 4% PFA/0.1% glutaraldehyde
for 20 minutes. The sections were dehydrated through a series of
alcohols and then counterstained with filtered 0.1% eosin in 95%
ethanol for 20 to 30 seconds. Rinses were made in 95% and then in
100% ethanol before transferral to histoclear and mounting in Vecta
mount (Vector Laboratories, Peterborough, UK).