1418 Veronica Basso et al. Eur. J. Immunol. 2011. 41: 1410–1419 mAbs for CD4, CD8, CD25, and CD44. Samples were collected on a FACSCANTO (Becton Dickinson) and then analysed using Flow Jo software. When indicated cells were labelled with the vital dye CFSE as described previously [39], stimulated with plate-bound anti-CD3 (2C11) and anti-CD28 (37.51) mAbs, and analyzed by ﬂow cytometry. Histology and immunohistochemistry Organs isolated from P13 mice were cryosectioned and processed for diaminobenzidine light or ﬂuorescence confocal immuno- cytochemistry as described previously [13, 40]. Sections were were stained with the indicated antibodies and counterstained with DAPI. Hematoxylin staining was performed on cryosections with Hematoxylin QS (Vector Labs). Sections were observed through an Ultraview (PerkinElmer) and a Zeiss upright microscope with Nomarski optics. Biochemical analysis Organs isolated from P9 or P13 mice were lysed with nine volumes of lysis buffer (0.5–1% Triton X-100, 150 mM NaCl, 20 mM Tris-Cl, pH 7.5, 1% sodium ﬂuoride, 1% sodium orthovanadate, cocktail of protease inhibitors). Cleared lysates were used for immunoblotting and immunoprecipitation with the indicated antibodies. For the detection of Rac3 in organs, lysates were incubated with anti-Rac3 antibody pre-adsorbed to protein A Sepharose beads, washed, and used for analysis by SDS-PAGE and immunoblotting with anti-Rac mAb recognizing all Rac proteins (Upstate Biotechnology). The ﬁrst antibodies were revealed either by 125I-protein A (Amer- sham) or by HRP-conjugated anti-rabbit or anti-mouse immu- noglobulins (ECL, Amersham). For the quantiﬁcation of the protein levels, bands from the immunoblots were analysed with a Personal SI Laser Densitometer (Molecular Dynamics, Sunnyvale, CA). Statistical analysis Data were analysed by the Student’s t test or by one-way ANOVA with Bonferroni correction: po0.05; po0.005; po0.001 comparing single or double KO with WT mice. Statistically signiﬁcant differences are indicated in the ﬁgures. Acknowledgements: We thank Emanuela Zuccaro and Roberta Pennucci for technical help, and Renata Mazzuchelli for reagents and suggestions. The support by Telethon-Italy (grant GGP09078 to IdC) and Ministero della Salute, Progetto & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Strategico RO/10/07 (RFPS-2006-3-337909) (to gratefully acknowledged. A.M.) is Conﬂict of interest: The authors declare no ﬁnancial or commercial conﬂict of interest. References 1 Wrona, D., Neural-immune interactions: an integrative view of the bidirectional relationship between the brain and immune systems. J. Neuroimmunol. 2006. 172: 38–58. 2 Kohm, A. P. and Sanders, V. 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