Publication | Open Access
An indirect immunofluorescence procedure for staining the same cryosection with two mouse monoclonal primary antibodies.
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1993
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Indirect Immunofluorescence ProcedureImmunocytochemical TechniqueMonoclonal Primary AntibodiesImmunologyPathologyImmunophenotypingImmunotherapyMyosin-specific MabImmunochemistryAutoantibodiesAllergyAutoimmune DiseaseHistopathologyAutoimmunityAntibody ScreeningCell BiologyMouse MabImmunoglobulin ESame CryosectionMonoclonal AntibodiesMedicineCryoglobulinemia
Indirect immunofluorescence methods have difficulty localizing two antigens in the same preparation when both primary antibodies are from the same species because secondary antibodies cross‑react. The authors present a protocol that sequentially stains a frozen muscle section with two mouse monoclonal antibodies against actin and myosin. They first apply a myosin‑specific mAb and detect it with a rabbit anti‑mouse IgG‑rhodamine secondary antibody, then block with a non‑binding mouse mAb and unconjugated goat anti‑mouse IgG F(ab) fragments, and finally apply an actin‑specific mAb detected with a rabbit anti‑mouse IgG‑fluorescein secondary antibody. Confocal microscopy shows clear identification of I‑bands, A‑bands, and H‑bands, and the method can be adapted to any pair of same‑species primary antibodies.
Indirect immunofluorescence procedures reported thus far are not effective at localizing two antigens in the same preparation when both primary antibodies are raised in the same species. In this case, the secondary antibodies can crossreact with both primary antibodies. We report here a protocol in which mouse monoclonal antibodies (MAb) specific for actin and myosin were used sequentially to stain the same frozen section of guinea pig skeletal muscle. The myosin-specific mAb was applied first and was localized with a rabbit anti-mouse IgG-rhodamine secondary antibody. The sections were then "blocked" with a non-binding mouse MAb and unconjugated goat anti-mouse IgG F(ab) fragments. The actin-specific mAb was then applied and localized with a rabbit anti-mouse IgG-fluorescein secondary antibody. Laser scanning confocal microscopy and image analysis demonstrated that the I-bands, the A-bands, and the H-bands of each sarcomere were clearly identifiable by this approach. This protocol is not limited to use with mouse MAb but can be easily modified to permit indirect immunolocalization of two antigens in the same sample using any pair of same-species primary antibodies.