Preparation of immunofluorescence double staining sample for laser confocal microscope

Objective To introduce the method of immunofluorescence double label for providing an ideal observation sample for laser scanning confocal microscope.MethodsRats were injected with Brdu intraperitoneally,and then fixed with perfusion of 40 g/L paraformaldehyde.The brain tissue of the rat was frozen and sliced in the frozen microtome.The slices was separately stained with polyclonal antibodies of anti-GFAP and monoclonal antibodies of anti-Brdu,and then,incubated with IgG labeled by FITC and Cy3 individually.A continuous layer scanning of the slices and image overlapping were performed under laser confocal microscope.ResultsRed fluorescence was dyed in the nucleus at the proliferative phase(S phase) and green fluorescence was shown in the cytoplasm of the astrocyte.ConclusionThere are many factors which will influence the result of immunofluorescence double staining.One of the most important factors is the arrangement and coordination in the pair of the first antibodies.