Eur. J. Immunol. 2011. 41: 1410–1419 Immunomodulation 1417 Figure 8. Suboptimal thymic innervation in Rac1N/Rac3KO double KO mice. (A) Cryosections of the thymus from P13 Rac1N/Rac3KO and control Rac3KO littermates were stained with anti-TH antibodies and analysed by confocal microscopy (representative images are shown in Supporting Information Fig. 7). The density of TH1 ﬁbres was quantiﬁed and expressed as the number of TH1 ﬁbres per 0.015 mm2 of thymic cortex. Data are mean7SEM of 80 ﬁelds from eight thymic sections per genotype from three independent experiments. Student t test: p 5 0.24 comparing control Rac3KO and Rac1N/Rac3KO littermates. (B–E) Tau and calnexin expression was evaluated by immunoblotting thymic lysates (100 mg protein per lane; for each comparison, two distinct pairs of littermates are shown) obtained at (B and C) P13 and (D and E) P9. (C and E) Expression of the major intermediate band of tau (tau-m) and of total tau (equal to the sum of the signals for all the bands speciﬁcally recognized by the anti-tau antibody) was normalized to that of calnexin and presented as mean7SEM (n 5 4–5 different thymi per mouse genotype). po0.05 by the Student’s t test comparing control Rac3KO and Rac1N/Rac3KO littermates. Materials and methods Mice Animal care was in accordance with institutional guidelines. Procedures for the generation of Rac1N, Rac3KO, and Rac1N/ Rac3KO mice, and for their genotyping have been described [14]. For experiments, Rac1F/F mice were crossed with Rac1F/1SynI- Cre mice to obtain Rac1F/F and Rac1F/FSynI-Cre littermates, deﬁned as WT and Rac1N respectively; whereas Rac1F/FRac3KO mice were crossed with Rac1F/1SynI-Cre/Rac3KO mice to obtain Rac1F/F/Rac3KO and Rac1F/FSynICre/Rac3KO littermates, deﬁned as Rac3KO and Rac1N/Rac3KO respectively. Antibodies The following antibodies were used for biochemical and morphological analysis: anti-calnexin polyclonal antibody (pAb) International, Temecula, CA); anti-b3 tubulin (Tuj1, from Berkeley Antibody Company, Richmond, CA); anti-neuroﬁlament (Afﬁnity); anti-TH (from Neuromics); anti-Rac3 pAb [13]; anti- Rac mAb (Upstate Biotechnology, Lake Placid, NY); anti-active caspase-3 mAb (BD Biosciences, San Jose, CA); anti-CD3e (clone CT-CD3; Valter Occhiena); anti-cytokeratin 5 (Covance); anti- cytokeratin 8 and anti-claudin-4 (Zymed), anti-FoxP3 (eBioscience), anti-TdT (DAKO). Flow cytometry analysis cells Mice were sacriﬁced at P4–P13. Thymus, peripheral lymph nodes, and spleen were collected, and homogenized into single-cell suspensions. Viable were enumerated by Trypan Blue-assisted counts. The cells were then incubated with a blocking buffer (5% rat serum and 95% culture supernatant of 2.4G2 anti-FcR mAb-producing hybridoma cells) for 20 min at 41C to saturate the FcRs. Cells were then stained for CD4, CD8, B220, CD44, CD62L mAb (BD Biosciences). (Sigma); anti-tau mAb, anti-choline acetyltransferase (Chemicon Thymocyte subpopulations were detected by staining with & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu