Simple method to induce denaturation of fluorescent proteins in free-floating brain slices

Abstract

Background: The generation of animals expressing reporter proteins (e.g., GFP, mCherry or tdTomato) under the control of genes of interest has become a valuable tool in neuroscience. However, the histological reuse of brain sections of these genetically modified animals in unplanned experiments is often infeasible since the constitutive expression of fluorescent reporter proteins interferes with further fluorescent staining procedures. Thus, expensive or time-demanding experiments frequently need to be repeated using additional experimental animals.

New method: To improve the reuse of tissues of reporter animals for fluorescent staining procedures, we developed fast, inexpensive and simple methods that induce denaturation of constitutively expressed fluorescent proteins in free-floating brain slices. These procedures consist of incubation of brain sections either in a 1% sodium hydroxide alkaline solution (pH 13.0) for one hour at room temperature or at 95 °C for 10-30 min.

Results: The strong fluorescence of tdTomato, mCherry and eGFP was completely eliminated after incubation of brain sections of different reporter mice in a pH 13.0 solution for one hour. hrGFP was resistant to denaturation in an alkaline solution, but incubation of brain sections at 95 °C for 10 min eliminated the fluorescence of hrGFP, as well as of tdTomato, mCherry and eGFP. The denaturing procedures did not prevent the reuse of brain tissues in free-floating immunofluorescence staining using multiple antibodies. Furthermore, the quality of the labeling remained unaffected. Although pretreatment in pH 13.0 solution maintained good tissue integrity, as a side effect, brain sections exhibited increased autofluorescence. However, a rinse in 0.25% Sudan Black B solution was efficient in eliminating the autofluorescence without impairing the immunofluorescence staining or DAPI counterstaining.

Conclusions: The present study provides simple procedures capable of inducing denaturation of fluorescent proteins in free-floating brain slices.

Keywords: Alkaline; Chemical denaturation; Cre-LoxP; GFP; Immunohistochemistry; TdTomato.