Fig. 1

Differential labelling of cerebellar cell types following EU injection. a Schematic representation of intracerebellar EU injection (midsagittal caudal mouse brain). b Widefield tile scan (b) and confocal images (b′, b″, 1-μm optical section) of coronal cerebellar sections of EU-injected mouse paraformaldehyde-fixed 10 min after termination of the injection. EU, visualized by click chemistry with Alexa555-azide, is widely distributed throughout the caudal vermis, with decreasing levels of EU in the lateral cerebellar hemispheres. EU-labelling fills the entire neuropil with slightly higher levels in the nuclei of Purkinje cells (asterisks in b′ and b″) and the walls of blood vessels (arrow in b′). c Widefield tile scans (c) and confocal images (c′, maximal projection of 20 images, 9-μm stacks) of cerebellar cortex of EU or saline injected animals perfused 1 h, 6 h or 24 h following EU injection. Note the absence of labelling in saline injected animals, and abundant nuclear EU labelling in all layers in EU injected animals. Further note, subtle differences in EU labelling at different post-injection time points, in particular in the granule cell layer (gl), where all cells are labelled at 1 h, while only a fraction of cells is labelled at 6 h and 24 h. d–h Confocal images (1-μm optical sections) of combined EU staining and immunofluorescence of cellular markers: Inositol trisphosphate receptor (IP3R), Purkinje cells; Neuronal nuclei antigen (NeuN), granule cells (arrow in d); Parvalbumin (Parv), molecular layer (ml) interneurons (arrows in g) and Purkinje cells; S100β, Bergmann glia (arrow heads in g) and other astrocytes; Iba1, microglia (h). i Widefield tile scan (i) and confocal image (i′, thickness optical section 9 μm) of EU labelling 1 h after EU-injection in a mouse that received an actinomycin D (ActD) injection 1 h prior to the EU injection in the same site. The yellow area (i) marks the area with either no or strongly reduced EU staining surrounding the ActD injection site. The arrows point to unlabelled (yellow) and weakly labelled (white) Purkinje nuclei. Scale bars: 500 μm (b, c), 50 μm (i′), 20 μm (b′, c, d, g) and 5 μm (b″, h, insert in d). 3, 6, 9 and 10, lobules 3, 6, 9 and 10 of cerebellar cortex; Cr2, Crus2 of cerebellar cortex; IC, inferior colliculus; MeO, medulla oblongata; PCl, Purkinje cell layer; wm, white matter