A universal method for sequential immunofluorescent analysis of chromatin and chromatin-associated proteins on chromosome spreads.

Immunofluorescence has been widely used to study histone modification dynamics and chromosome-associated proteins that regulate the segregation of chromosomes during cell divisions. Since many of these regulatory proteins interact (in)directly to exert their proper function, it is of interest to detect these proteins simultaneously, to establish their spatiotemporal relation. However, the detection of multiple epitopes on the same material is limited by the availability of antibodies derived from different host species. For Western blot membranes, buffers were developed to remove antibodies after the first round of detection and enable a second round of detection. In this study, we establish that this "stripping" principle can also be applied for sequential immunofluorescence on chromosome preparations. We first adapted a drying down fixation technique for the use on cultured cells from different primary cells and cell lines. These chromosome spreads were subsequently used to optimize the stripping procedure for this application. We investigated feasibility and reliability of detection of histones and their posttranslational modifications as well as chromatin interacting proteins in two subsequent rounds of immunofluorescence. We conclude that this method is a reliable option when spatial resolution and co-expression need to be investigated and the material or the choice of antibodies is limited.