Click histochemistry for whole-mount staining of brain structures

Abstract

Labeling of the replicating DNA with synthetic thymidine analogs is commonly used for marking the dividing cells. However, until now this method has only been applied to histological sections. A growing number of current approaches for three-dimensional visualization of large tissue samples requires detection of dividing cells within whole organs. Here we describe a method for labeling dividing cells with 5-ethynyl-2'-deoxyuridine (EdU) and their further detection in whole brain structures (for example, hippocampus) using the Cu (I) -catalyzed [3 + 2] cycloaddition reaction (so-called click-reaction). The presented method can be used for brain neurogenesis studies as well as for whole-mount staining of any preparations in which the terminal ethynyl group has been introduced. •New click histochemistry method based on Cu (I) -catalyzed [3 + 2] cycloaddition reaction allows whole-mount staining of brain structures and other tissues.•Our whole-mount click histochemistry method allows to visualize dividing cells in 3D and can be used in neurogenesis studies, i.e. for birthdating dividing early progenitors and further tracking of proliferation, survival, migration, differentiation, and fate of their progeny.•Our whole-mount click histochemistry staining demonstrates high staining specificity, high signal intensity, and low background levels in young and adult mouse brain tissue.

Keywords: 3D imaging; 5-ethynyl-2'-deoxyuridine; Cell division; Cell migration; Click chemistry; Hippocampus; Neurogenesis; Whole-mount click histochemistry; Whole-mount histochemistry.

Graphical abstract

Fig. 1

Dividing cells in whole hippocampi of 2-week-, 2-month- and 12-month-old mice. A,D,G and B,E,H – 3D reconstructions; C,F,I – optical sections. Arrows in A,D,G and C,F,I show the dentate gyrus; B,E,H - enlarged fragments of areas marked by arrows in A,D,G. Scales are 200 μm.