Fig. 3. Recombination analysis in the vermis of En2cre; Rb^LoxP/LoxP mice. (A,B) Whole-mount ß-galactosidase staining of the vermis of En2cre, ROSA26^LoxP indicator mice shows the area of recombination from outside (A) and after sagittal sectioning (B). (C) Histology of an area corresponding to the red square in B (Nuclear Red counterstaining; EGL, external granular layer; PC, Purkinje cells; IGL, internal granular layer), demonstrating the lacZ expression in all areas of the cerebellum. Adjacent sections were immunostained for calbindin (D) or NeuN (E) to confirm the En2cre-mediated recombination in these cell populations. (F-I) PCR analysis of cell fractions obtained from Percoll gradients. Fraction 1 contains larger cells (astrocytes, GFAP immunostaining in F; Purkinje cells, Hematoxylin and Eosin staining in G), while Fraction 2 contains small cells, such as granule cells and granule cell precursors (Hematoxylin and Eosin staining in H). (I) PCR recombination analysis of both fractions shows a 283 bp product representing the floxed allele (primers Rb19E and Rb18) and a 260 bp product of recombined Rb allele (primers Rb212 and Rb18). Genomic DNA extracted from Percoll-separated cerebellar fractions of Rb^LoxP/LoxP and En2cre;Rb^LoxP/LoxP mice were used in lanes 3,4 and 5,6, respectively. Lanes 3 and 5, larger size fraction 1. Lanes 4 and 6, lower size fraction 2. Two controls with partial recombination are shown in lanes 1 and 2.