Incubate in a humidified chamber overnight at 4✬.Add primary antibodies (diluted in 1x PBS, containing 0.3% Triton, 0.01% NaAzide, 0.02% Bacitracin), approx.Block sections in 2% normal serum of the secondary antibody host, in PBS 30 min at RT (optional).Paraffin sections: Put slides in 0.3% H2O2 in 1xPBS for 15 min at RT to quench endogenous peroxidase activity.Cryosections: Put slides in 0.03-0.1% H2O2 in 1xPBS for 5 min at RT to quench endogenous peroxidase activity.Rinse slides in 1x wash buffer for 15 min.Draw a circle around each section with DAKO Cytomation pen.If primary antibodies are of different isotypes (IgG1, IgG2a, IgG2b), they can be mixed together. Optimal dilutions must be determined by the user. NOTE: The specified working dilutions of the primary antibodies are to be considered as a guideline only. Rehydrate sections: 100% EtOH 2x3 min, 95% EtOH 2x3 min, 70% EtOH 2x3 min, rinse in ddH20.Deparaffinize sections in xylene (2x5 min).HIER buffer pH 9, a Tris/EDTA buffer, 97☌, 20 min.Ĭool down with ddH2O, rinse in 1x wash buffer 15 min, and proceed with IHC.HIER buffer pH 6, a modified citrate buffer, 97☌, 20 min.HEIR buffer pH 9, a Tris/EDTA buffer, 60-70☌, 10 min.Īntigen retrieval for paraffin sections Heat-Induced Epitope Retrieval (HIER).HIER buffer pH 6, a modified citrate buffer, 60-70☌, 10 min.If the target requires antigen retrieval, standard target retrieval solutions can be used, though the temperature and time should be reduced. Antigen retrieval for cryosections (optional) Perfusion- or immersion-fixed paraffin-embedded sections cut at 4 μm thick, mounted on Super Frost slides, dried at 50☌ overnight. Pre-fix sections in ice-cold paraformaldehyde (4% PFA) for 20 min (put frozen sections directly into 4% PFA, do not allow thawing). Snap-frozen: Sections cut in a microtome at 14 μm, thaw-mounted on Super-Frost slides and dried for 2 h. Rehydrate in 1xPBS 2x15 min, or proceed with antigen retrieval. Dry sections on slides for an additional 2 h at room temperature (RT). Perfusion-fixed: 10-30% sucrose cryoprotected sections cut in a microtome at 14 μm, thaw-mounted on Super Frost slides. Pre-incubate primary antibody with BSA (0.5%) prior to application to the tissue.ĭilute primary antibody in antibody diluent to a working concentration. Eppendorf tubes for dilution of antibodies.Fluorophore-conjugated secondary antibodies for regular immunofluorescence.Primary and secondary antibodies diluted in 1x PBS, containing 0.3% Triton, 0.01% NaAzide, 0.02% Bacitracin and BSA.Incubation chamber with wet Wettex stripes.Target retrieval solutions pH6 or pH9 (if needed).Cryo- or paraffin-embedded sections mounted on SuperFrost slides.Moreover, several control experiments must be performed to ensure the specificity of co-localisation of different targets (see below). Many companies have special antibodies with minimal cross-reactivity developed especially for co-localisation studies. The secondary antibodies should be from the same species (to prevent cross-reactivity). primary antibodies of different isotypes, which can then be visualized using subtype-specific secondary antibodies. In the latter case, it is possible to use e.g. Primary antibodies should ideally be from different species, but in some cases, it is possible to use antibodies raised in the same species. Multiplex immunofluorescence allows the simultaneous detection of several target proteins in the same cell.
#CONTROLS FOR ANTIBODY CROSS REACTIVITY PDF#
Download a pdf version of the the IHC-IF Multiplex protocol.
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