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

Author(s)

Moinho, Tais M. ; Tavares, Mariana R. ; Campos, Ana M. P. ; Frazao, Renata ; Metzger, Martin ; Donato, Jose, Jr.

• Document type: Journal article
• Source: JOURNAL OF NEUROSCIENCE METHODS; v. 371, p. 10-pg., 2022-02-15.

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 degrees 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 degrees 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. (AU)

• FAPESP's process: 19/21707-1 - Food deprivation effects in kisspeptin neurons excitability
• Grantee: Renata Frazão
• Support Opportunities: Regular Research Grants
• FAPESP's process: 20/01318-8 - Central nervous system as a target of growth hormone for the regulation of multiple biological functions
• Grantee: Jose Donato Junior
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 20/10102-9 - Study of the effects induced by GH receptor ablation in neurons of the lateral hypothalamic area
• Grantee: Mariana Rosolen Tavares
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 17/16473-6 - Processing of aversive stimuli: circuitry between the laterodorsal tegmental nucleus, the habenula, and dorsal and median raphe nuclei
• Grantee: Martin Andreas Metzger
• Support Opportunities: Regular Research Grants