Quantitative comparison of DNA detection by GFP-lac repressor tagging, fluorescence in situ hybridization and immunostaining

Background: GFP-fusion proteins and immunostaining are methods
broadly applied to investigate the three-dimensional organization
of cells and cell nuclei, the latter often studied in addition by
fluorescence in situ hybridization (FISH). Direct comparisons of
these detection methods are scarce, however. Results: We provide a
quantitative comparison of all three approaches. We make use of a
cell line that contains a transgene array of lac operator repeats
which are detected by GFP-lac repressor fusion proteins. Thus we
can detect the same structure in individual cells by GFP
fluorescence, by antibodies against GFP and by FISH with a probe
against the transgene array. Anti-GFP antibody detection was
repeated after FISH. Our results show that while all four signals
obtained from a transgene array generally showed qualitative and
quantitative similarity, they also differed in details. Conclusion:
Each of the tested methods revealed particular strengths and
weaknesses, which should be considered when interpreting respective
experimental results. Despite the required denaturation step, FISH
signals in structurally preserved cells show a surprising
similarity to signals generated before denaturation.