Antibody incubations are performed in one of two buffer systems. One buffer consists of 1X PBS containing 2 mg/mL BSA and 0.1% Triton X-100 (PBT). The other is a maleic acid buffer developed by Tabitha Doniach (MAb = 100 mM maleic acid, 150 mM NaCl, pH 7.5). If background staining becomes a problem, the MAb is recommended. After the final wash in PBST, proceed as follows:
1. Wash three times for 5 min with PBT or MAb at room temperature.
2. Block tissues by incubating one hour at room temperature in PBT + 20% Heat-treated lamb serum. Lamb serum is heat-treated by placing it in a water bath at 55-60°C for 30 min. The serum is then stored frozen in aliquots. Alternatively, MAb containing 2% blocking reagent from Boehringer Mannheim (cat. no. 1096 176) and 20% serum can be used.
3. After blocking, replace the blocking buffer with the same solution containing a 1:2000 dilution of antidigoxygenin antibody (Fab fragments) coupled to alkaline phosphatase (BMB cat. no. 1093 274). Incubate overnight at 4°C, or at least 4 h at room temperature with constant shaking.
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