Objective Detailed descriptions of the double-labeling immunofluorescence staining for fluorescence microscopy provides an ideal sample.Methods Rat liver frozen sections were used fixative were 95％ alcohol,95％ formaldehyde and acetone,frozen sections,with anti-CSE,Ki-67 polyclonal antibody and incubated with FITC,Cy3 fluorescence-labeled secondary fluorescently labeled secondary antibody staining,observed under a fluorescence microscope.Results Acetone fixed group visible in the proliferative phase (S phase) cells showed a red fluorescent nucleus,cytoplasmic green fluorescence.Conclusion The impact of double labeling immunofluorescence the effect of sample links and many factors,including the two most important factors are 2 and coordination with the primary antibody and select the appropriate fixative.