2. Primary antibody (as determined by the experimental aim).
3. PBST: PBS with 0.4% Triton X-100. Store at 4°C.
4. Antibody dilution buffer: PBST containing 1% NGS. Prepare fresh.
5. Secondary antibody (select appropriate detection tag as well as specificity for the primary antibody species being used) diluted in PBST with 1% NGS.
For horseradish peroxidase (HRP) detection: We use affinity purified HRP-
conjugated goat anti-IgG (heavy and light chain specific) antibody raised against either mouse (Bio-Rad, no. 170-6516) or rabbit (Bio-Rad, no. 1706515). These antibodies will detect both IgGs and IgMs.
For fluorescent detection we generally use affinity purified antibodies from either Cappell (ICN) or Jackson ImmunoResearch conjugated to either Texas Red, tetramethylrhodamine isothiocyanate (TRITC), fluorescein isothiocyanate (FITC), or cyanine 5 (Cy5). For multiple labelings using monoclonal antibodies, a variety of isotype-specific antibodies are available.
7. 3,3'-Diaminobenzidine tetrahydrochloride (DAB) stock solution: 10 mg/mL DAB in 0.05 M Tris-HCl, pH 7.5. (DAB is a suspected carcinogen; avoid contact, inhalation, or ingestion.) Store at -20°C protected from light.
8. H2O2 (hydrogen peroxide). (Toxic; avoid contact, inhalation, or ingestion.) Store at 4°C.
9. PBS containing 0.05% sodium azide.
10. Acrodisc syringe filters (Fisher, no. 09-730-218).
11. Aluminum foil.
12. Hoechst 33258 (Molecular Probes, no. H-3569), 0.2 ^g/mL in PBS (Possible mutagen; avoid contact, inhalation, or ingestion.) Store at 4°C.
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