This protocol assumes that the first and second primary antibodies have been raised in different hosts. If this is not the case, see Note 9. Fluorescent labels for the secondary antibodies should be chosen to the capabilities of the microscope that will be used.
The protocol specifies staining serially for two antigens. Serial staining offers a great deal of flexibility, which is particularly useful when still experimenting with a stain. Conditions can be varied for each antibody; the sample can be divided and stained differently after the first staining, or postfixed, and so on. The downside is that it takes longer, and during the extra steps, there is more opportunity for dissociation of antibody-antigen complexes and even reversal of crosslinks in the fix. Postfixation can prevent this problem but may affect staining for some antibodies.
Antibodies are diluted in the wash solution. All blocking, incubation, and clearing steps are done in 0.5-mL eppendorf tubes on a rotator. Storage between steps should be at 4°C in the wash solution because formaldehyde crosslinks are not stable in aqueous solutions and there can be exchange between antibodies and antigens. Try to minimize the length of time between steps; for stainings that take more than 1 d, do one of the washes at 4°C overnight.
1. Block and permeabilize in block-permeabilization solution for 1-2 h at room temperature or 4 h at 4°C (see Note 10).
2. Incubate with host 1 primary antibody 1 (e.g., rabbit anti-lamin) in Wash solution for 1-2 h at RT (see Note 11).
3. Clear by washing three times with the wash solution for 20 min each (a minimum of 60 min for all washes). If using a labeled primary antibody, skip steps 4 and 5.
4. Incubate with anti-host 1 secondary antibody in the wash solution for 1-2 h at RT.
5. Clear by washing three times with Wash solution for 20 min each (a minimum of 60 min for all washes). If using a labeled primary antibody, skip steps 6 and 7.
6. Incubate with anti-host 2 secondary antibody in the wash solution for 1-2 h at RT.
7. Clear by washing three times with the wash solution for 20 min each (a minimum of 60 min for all washes).
8. Optional: If you are not already using a fluorophore that excites in the ultraviolet (UV) range and have a DAPI filter, incubate with DAPI (1 |g/mL final concentration) for 10 min. It is a good way to locate cell nuclei.
9. Final clear by washing three times with the final clearing solution for 20 min each (a minimum of 60 min for all washes). The substitution of Brij35 (which does not fluoresce) for Triton X-100 and the removal of the BSA should reduce background (see Note 12 for staining controls for single- and multiple-label staining).
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