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Slides for IHC were then incubated for 10 minutes in 0.5% hydrogen peroxide in methanol to quench endogenous peroxidases, however this step was omitted for IF slides.
Immunofluorescence (IF) slides were counterstained with the nuclear dye DAPI.
For IF, slides were incubated with anti-rabbit Cy3 and anti-mouse FITC (Molecular Probe, Eugene, OR, USA) antibodies at 1 1,000 in 4% BSA/PBST.
For immunofluorescence (IF), slides were deparrafinized with xylene, rehydrated with ethanol to 1× PBS washes, and boiled at 97 °C for 16 min in 1 M Urea (Bio-Rad, 161-0730) or 1× Vector Unmasking Solution (TNFSF11 only).
The problem may be magnified if slides from local institutions are shipped to central laboratories for immunohistochemistry.
Ambitious research projects that have not been adequately organized may suffer the consequences of poor quality or unreadable stains if slides are not used relatively quickly after sectioning.
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